Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(2): 99-110, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-573 Citation: R. Tabaripour, M. Sheidai, S. Mehdi Talebi, Z. Noormohammadi (2020) Population genetic and phylo- geographic analyses of Ziziphora clino- podioides Lam., (Lamiaceae), “kakuti-e kuhi”: An attempt to delimit its subspe- cies. Caryologia 73(2): 99-110. doi: 10.13128/caryologia-573 Received: July 26, 2019 Accepted: April 16, 2020 Published: July 31, 2020 Copyright: © 2020 R. Tabaripour, M. Sheidai, S. Mehdi Talebi, Z. Noormo- hammadi. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Population genetic and phylogeographic analyses of Ziziphora clinopodioides Lam., (Lamiaceae), “kakuti-e kuhi”: An attempt to delimit its subspecies Raheleh Tabaripour1,*, Masoud Sheidai1, Seyed Mehdi Talebi2, Zahra Noormohammadi3 1 Faculty of Biological Sciences and biotechnology, Shahid Beheshti University, Tehran, Iran 2 Department of Biology, Faculty of Science, Arak University, Arak, Iran 3 Biology Department, Islamic Azad University, Sciences and Research Branch, Tehran, Iran *Correspondence author. E-mail: raheleh.tp@gmail.com Abstract. Ziziphora clinopodioides Lam., (Lamiaceae), is a perennial herb which is used as traditional medicine in Iran. Different authors disagree on the number of subspe- cies. In general, taxonomic and biosystematic studies of Ziziphora clinopodioides have been limited and no molecular phylogenetic or biogeographic study of the species has been carried out. Therefore, the aims of this study were (1) to determine the number of subspecies, (2) to produce information on the species’ genetic structure and intra-spe- cific genetic variability, and (3) to produce data on the probable date of appearance of Ziziphora clinopodioides in Iran. We used a combination of morphological and molecu- lar data to study plants randomly collected from 5 geographical regions. Both analyses revealed a high level of within population variability and grouping of the studied prov- inces produced an admixture that indicated the absence of any subspecies within the species. STRUCTURE analysis and K-Means clustering identified two gene pools with- in the country. The probable date of divergence obtained was 5-10 Mya for the appear- ance of this species in the mountainous regions of Qazvin and Mazandaran. Keywords: biogeography, genetic diversity, STRUCTURE analysis, subspecies delimi- tation, Ziziphora clinopodioides. INTRODUCTION Species and subspecies delimitation is a difficult and somewhat subjec- tive task in a species complex and in species with several overlapping geo- graphical populations (Wiens 2007). In general, the recognition of a species or subspecies is based on morphological observations. Different species can be delimited by a few distinct morphological char- acteristics that show no overlap with other species. This criterion is very tra- 100 Raheleh Tabaripour et al. ditional yet it makes sense biologically, which suggests that there is no gene flow between the species (based on some assumptions; for example any morphological difference has a genetic basis) (Wiens 2007). However, this approach can either fail to discriminate species and mask the presence of cryptic species or discriminate dif- ferent species while in reality there is only one. In these situations, it is suggested that different and combined approaches such as morphological, molecular, cytologi- cal, and other approaches are used to determine species boundaries (Duminil and Di Michele 2009; Carstens et al. 2013). In some cases, incongruence may occur across the results from different methods. This may be due to either introgression or a difference in the power to detect cryptic lineages across one or more of the approaches used (Carstens et al. 2013). In recent years, parallel developments in the ana- lyzing power of both molecular phylogenetic and popu- lation genetic methods as well as their use in combina- tion have resulted in more powerful species delimita- tion strategies. One of the most striking examples of a joint population genetics and phylogenetic approach is the use of the multispecies coalescent model to estimate phylogeny (Edwards 2009; Kingman 1982). This is fur- ther strengthened by development of new algorithms for detecting population genetic structure (Pritchard et al. 2000; Huelsenbeck and Andolfatto 2007; Huelsenbeck et al. 2011). The procedure usually involves comparing clusters obtained on the basis of observed polymorphism in both morphological and molecular characters to test if they are in agreement. In case of infra-specific taxon identi- fication (e.g. subspecies), the occurrence of discontinu- ity in both datasets can be suggestive (Seif et al. 2012; Koohdar et al. 2015). K nowles and Carstens (2007) addressed how molecular data (i.e., gene trees from DNA sequence data) can be used in species delimitation. They pro- posed a new method which uses coalescent simulations to test hypotheses about species limits. Their method is particularly valuable in that it can incorporate data from multiple loci and does not require species to have diverged to the point of being reciprocally monophyl- etic. Similarly, Medrano et al. (2014), applied population genetics methods to the species delimitation problem in Narcissus (Amaryllidaceae) using amplified fragment length polymorphism (AFLP) molecular markers. Ziziphora clinopodioides Lamarck (Lamiaceae) is a perennial herb with the common Persian name “kakuti- e kuhi”. It is used as a traditional medicine in Iran to treat diseases such as the common cold, gastrointesti- nal disorders and inflammation (Naghibi et al. 2010). Controversy exists as to the number of subspecies that should be recognized. For example, there are nine sub- species native to Iran according to Flora Iranica (Rech- inger 1982), but in the Flora of Iran (Jamzad 2012), no subspecies are considered. Ziziphora clinopodioides has prostrated to erect stems and mainly branches at the base. The leaves vary in size and shape. The flowers are light to dark pur- ple and white, with or without a peduncle, gathered in a compact capitulum. It is distributed in the eastern Balkan Peninsula, south east Asia and central Asia to the Pamir-Altay mountains and the Himalayas (Iran, Iraq and central and eastern parts of Turkey) as well as in Africa (Beikmohammadi 2011). In Iran it grows on rocky slopes, low hills and grasslands. In general, there has been no detailed study looking at the taxonomy, molecular phylogeny and biogeography of this species. Therefore, the aims of this study were (1) to determine the number of subspecies, (2) to produce information on the species genetic structure and intra- specific genetic variability and (3) to produce data on the probable date of appearance in Iran of Z. clinopodioides and its ancestral area of distribution. MATERIAL AND METHODS Plant materials In the present study, 69 plant specimens from 19 populations of Z. clinopodioides were randomly collected from five geographical localities (five provinces) of Iran. These populations occur from northern to eastern parts of the country and have almost a continous pattern of distribution. (Table 1, Fig. 1). Voucher specimens are deposited in the Herbarium of Shahid Beheshti Univer- sity (HSBU). Morphological studies In total 29 morphological (5 qualitative, 24 quanti- tative) characters were studied. These characters include both vegetative and reproductive (f loral) variables (Tables 2, 3). Molecular studies For molecular analyses, we used both multilocus molecular markers of inter-simple sequence repeats (ISSRs) as well as the chloroplast rpL16 region. For ISSR analysis we used 69 specimens (1-6 samples from each 101Population genetic and phylogeographic analyses of Ziziphora clinopodioides population) and for cpDNA analysis, we used a subset of 15 randomly selected plants (1-3 samples) from the stud- ied populations of five provinces (Table 1). Both markers are widely used for species diversity analysis and phylogeny (Weising et al. 2005; Sheidai et al. 2014). ISSRs are particularly suitable markers for infra-spe- cific studies and can reveal genetic discontinuities among populations (Sheidai et al. 2012; Sheidai et al. 2013). DNA extraction, amplification and ISSR assay Genomic DNA was extracted using a CTAB (cetyl trimethyl-ammonium bromide) activated charcoal pro- tocol (Sheidai et al. 2013). The quality of extracted DNA was examined by running on a 0.8% agarose gel. 10 ISSR (inter simple sequence repeat) primers, (AGC)5GT, (CA)7GT, (AGC)5GG, UBC810, (CA)7AT, (GA)9T, UBC807, UBC811, (GA)9A and (GT)7CA, were used (University of British Columbia). PCR reac- tions were performed in a 25μl volume containing 10 mMTris-HCl buffer at pH 8, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP (Bioron, Germany), 0.2 μM of each primer, 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany). The reactions were performed in a Techne thermocycler (Germany) with the following program: 5 min initial denaturation step at 94 °C, followed by 40 cycles of 45s at 94 °C; 1 min at 60 °C and 1min at 72 °C. The reaction was completed with a 7 min extension step at 72 °C. The amplification products were visualized by run- ning on 2% agarose gels. The fragment size was esti- mated using a 100 bp molecular size ladder (Fermentas, Germany). In order to identify reproducible bands, the experiment was replicated 3 times. Table 1. Locality information for populations of Z. clinopodioides sampled, including herbarium vouchers for specimens used for morpho- logical and ISSR analyses and GenBank Accession numbers for specimens used for cp-DNA analysis. Pop no. Province Elevation (m) Longitude Latitude Number of specimens sampled Voucher No. GenBank Accession no.ISSR & Morphology cpDNA 1 Razavi Khorasan 2042 352715.9 595344.9 4 1 HSBU2014413  (1) MG738475 2 Razavi Khorasan 1976 353559.4 5839.7 4 2 HSBU2014414 (2) MG738476 HSBU2014427 (3) MG738477 3 Mazandaran 1039 363610.3 534952.7 4 3 HSBU2014415 (4) MG738478 HSBU2014428 (10) MG738484 HSBU2014429 (11) MG738485 4 Mazandaran 2597 368309 511855 6 2 HSBU2014421 (5) MG738479 HSBU2014430 (6) MG738480 5 Tehran 2978 354349 521384.8 4 1 HSBU2014425 (7) MG738481 6 Qazvin 1400 362765.5 501711.4 5 2 HSBU2014431 (8) MG738482 HSBU2014432 (9) MG738483 7 Mazandaran 2225 363107 5456 3 2 HSBU2014426 (12) MG738486 HSBU2014433 (13) MG738487 8 Ardebil 1493 381209.9 483909.2 6 1 AUH522 (14) MG738488 9 Ardebil 1389 381235 481757.3 3 1 ALUH526 (15) MG738489 10 Tehran 2308 355775.2 512954.5 2 HSBU2014424 11 Qazvin 2750 392936 573450 4 HSBU2014416 12 Qazvin 1333 363155.1 509824 5 HSBU2014417 13 Razavi Khorasan 2652 362319.7 5959.5 6 HSBU2014412 14 Mazandaran 2103 362633.5 512838 1 HSBU2014419 15 Mazandaran 2299 355514 521172.8 2 HSBU2014420 16 Mazandaran 2341 355293.4 528320 4 HSBU2014423 17 Mazandaran 1510 311137.1 523006.6 1 AUH529 18 Tehran 3245 362229.8 512628.2 3 HSBU2014422 19 Tehran 2398 354636.4 515869.6 2 HSBU2014418 102 Raheleh Tabaripour et al. Chloroplast DNA The intron in the gene for ribosomal protein L16 (rpL16) located in the chloroplast genome was ampli- f ied and sequenced with universal primers follow- ing the methodology of Shaw and Small (2005) and Timmer et al. (2007). The rpL16 forward primer was 5´-GTAAGGGTCATTTAGTAGGTCGTTT -3´ and the reverse primer 5´-TCCTTACCATTAAGTTGATC -3 .́ Each 20 µl PCR tube contained 10 µl of 2x PCR buffer, 0.5 mM of each primer, 200 mM of each dNTP, 1 Unit of Taq DNA polymerase (Bioron, Germany), and 1 µl of template genomic DNA at 20 ng µl-1. The amplification reaction was performed in a Techne thermocycler (Ger- many) with the following program: 2 min initial dena- turation step at 94°C, followed by 35 cycles of 5 min at 94°C; 1.30 min at 62°C and 2 min at 72°C. The reac- tion was completed by a final extension step of 7 min at 72°C. PCR products were visualized on 2.5% agarose gels with GelRed™ Nucleic Acid Gel Staining. Fragment siz- es were estimated using a 100 bp size ladder (Thermo- Fisher Scientific, Waltham, MA USA). Data analyses Morphometry Morphological characters were first standardized (Mean = 0, Variance = 1) and used to establish Euclid- ean distances among pairs of taxa (Podani 2000). For grouping of the plant specimens, the UPGMA (Unweighted pair Group Method with Arithmetic Mean) and ordination method of PCA (principal components analysis) were used (Podani 2000). A PCA (principal components analysis) biplot was used to identify the most variable morphological characters among the stud- ied populations (Podani 2000). PAST version 2.17 (Ham- mer et al. 2012) was used for multivariate statistical analyses of morphological data. Table 2. Qualitative morphological characters studied in Z. clinopo- dioides populations. Character State of character and their codes Vegetative form Straight (1), Geniculate (2) Basal vegetative form Woody (1), Dense woody (2), Sparse woody- stacked (3) Stem leaf shape Lanceolate (1), Lanceolate-ovate (2), Multiform (3) Calyx hair frequency Frequent (1), Sparse (2), Very sparse (3) Calyx pedicle Present (1), Not present (2) Table 3. Quantitative morphological characters studied in Z. clino- podioides populations. No. Characters 1 Plant length (cm) 2 Leaf length of stem(mm) 3 Leaf width of stem(mm) 4 Stem Leaf length / width ratio 5 Petiole length(mm) 6 Inflorescence leaf length (mm) 7 Inflorescence leaf width (mm) 8 Inflorescence leaf length/ width ratio 9 Pedicle length (mm) 10 Calyx length(mm) 11 Calyx width(mm) 12 Calyx length/ width ratio 13 Calyx teeth length(mm) 14 Calyx teeth width(mm) 15 Calyx teeth length/ width ratio 16 Inflorescence length(cm) 17 Inflorescence width(cm) 18 Inflorescence length/ width ratio 19 Corolla length(mm) 20 Corolla tube length(mm) 21 Petal length(mm) 22 Corolla tube length/Petal length 23 Stamen length(mm) 24 Style length(mm) Figure 1. Distribution map of the studied provinces. 103Population genetic and phylogeographic analyses of Ziziphora clinopodioides ISSR analyses ISSR bands obtained were coded as binary char- acters (presence = 1, absence = 0). For grouping of the studied provinces, ISSR bands obtained were coded as binary characters (presence = 1, absence = 0). For group- ing of the studied provinces, PCO plot (principle coordi- nate analyses) was used (Noormohammadi et al. 2011). The Mantel test was performed to check correla- tion between geographical distance and genetic distance of the studied provinces (Podani 2000). The PAST ver. 2.17 (Hammer et al. 2012) program was used for these analyses. AMOVA (Analysis of molecular variance) based on Fst and Nei’s Gst as implemented in GenAlex 6.4 (Peakall and Smouse 2006) was used to reveal genetic difference of the studied provinces. In order to deter- mine the genetic structure of geographical provinces, we used two different approaches. First, Bayesian mod- el based STRUCTURE analysis (Pritchard et al. 2000), and second, the maximum likelihood based method of K-means clustering. For STRUCTURE analysis with 105 permutations, data were scored as dominant mark- ers (Falush et al. 2007). We performed K-means cluster- ing in GenoDive ver. 2. (2013). Two summary statistics, 1) pseudo-F, and 2) the Bayesian Information Criterion (BIC), provide the best fit for k in the K-Means cluster- ing method (Meirmans 2012). The population assignment test was performed using the maximum likelihood method as implemented in GenoDive (Meirmans and Van Tienderen 2004). In order to identify agreement between the genetic tree and the morphological tree, we obtained a consen- sus tree using DARwin ver.5 (2012). cp-DNA sequence analyses and estimation time of diver- gence The intron in the gene for ribosomal protein L16 (rpL16) was aligned with MUSCLE (Robert, 2004) implemented in MEGA 5. The molecular clock test was performed as implemented in MEGA 5 (Tamura et al. 2011). The test was done by comparing the ML value for the given topology with and without the molecular clock constraints under the Tamura and Nei (1993) model. using the parsimony method of Templeton et al. (1992), implemented in TCS 1.13 program (Clement et al. 2000). Before estimating time of divergence, we used MEGA 5 to test the molecular clock and to find the best substi- tution model for the given sequences. The equal evolu- tionary rate of the studied sequences was rejected at a 5% significance level and therefore we used the relaxed molecular clock model in further analyses (Drummond et al. 2006). Moreover, HKY was the best substitution model identified by model test as implemented in MEGA 5 (Posada and Crandall 1998). BEAST v1.6.1 (Drummond et al. 2010a; Drummond et al. 2010b) was used for the Bayesian MCMC inferred analyses of the nucleotide sequence data (Drum- mond and Rambaut 2007). Lallemantia baldschuani- ca Gontscharow, L. iberica Fisch. & C.A. Mey. and L. royleana Bentham were used as outgroups. BEAUti (Bayesian Evolutionary Analysis Utility ver- sion) v1.6.1 (Drummond et al. 2010a, 2010b) was utilized to generate initial xml files for BEAST. A Yule process of speciation (a ‘pure birth’ process) was used as a tree prior for all the tree model analyses. The Yule tree prior is widely recognized as giv- ing the best-fit model for trees describing the relation- ships between different species (Drummond et al. 2010a, 2010b) and can be regarded as explaining the net spe- ciation rate (Nee 2006). For the MCMC analyses, the chain length was 10000000. After discarding 100 trees representing the burn-in, 10000 trees were used for the analyses. The BEAUti xml file was run in BEAST v1.6.1 (Drummond et al. 2010a, 2010b). Because no fossils are available for the studied species, we assumed a rate of evolution of the plastid sequence (u = 1.0 X 10 -9 s s-1 year-1) (Zurawski et al. 1984; Minaeifar et al. 2016). This was included in the option of molecular clock model in BEAUti v1.6.1. The normal distribution (Mean = 0, Standard deviation = 1) was used for priors. Tracer v1.5 (Drummond and Rambaut 2007) was used to examine sampling and convergence. Tree Anno- tator v1.6.1 (Drummond and Rambaut 2007) was used to annotate the phylogenetic results generated by BEAST to form a single ‘target’ tree (Maximum Clade Credibility tree, MCC) including summary statistics. FigTree v1.3.1 (Rambaut 2009) was used to produce the annotated BEAST MCC tree (Fig. 6). Biogeography The distribution range of Ziziphora clinopodioides studied was divided into 5 areas (provinces): A (Razavi Khorasan), B (Ardebil), C (Mazandaran), D (Qazvin) and E (Tehran). We used S-DIVA (Statistical Dispersal- Vicariance Analysis) and BBM (Bayesian Binary Meth- od) analyses implemented in RASP to reconstruct the possible ancestral ranges on the phylogenetic trees (Yu et al. 2010, Yu et al. 2015). In these methods, the frequen- cies of an ancestral range at a node in ancestral recon- structions are averaged over all trees (Yan et al. 2010). We used initially the tree obtained from the BEAST 104 Raheleh Tabaripour et al. analysis (MCC tree), followed by RASP analysis. The final tree for the area ancestry determination was based on the majority rule consensus tree. RESULTS Systematics Morphometry The mean values and standard errors for the quanti- tative morphological characters are provided in Table 4. The ANOVA test revealed significant difference in stem leaf length (p = 0.01), inflorescence leaf length / width ratio (p = 0.01) and corolla tube length/petal length ratio (p = 0.02). Different clustering and ordination methods pro- duced similar results, therefore only the PCA plot of the studied provinces based on the morphological data is provided (Fig. 2). The studied provinces were placed inter-mixed, thus there is no support for morphological divergence among provinces. There appears to be some morphological differentia- tion between province 2 (Razavi Khorasan) and all other provinces which is plausible as it is the most geographi- cally separated (Fig. 2). PCA analysis of morphological characters revealed that the first three PCA components comprised 70% of the total variability. Morphological traits (stem leaf shape, petiole length and style length) showed the highest level of correlation with the first PCA component (>0.65), while characters 6 and 7 were highly correlated with the second PCA component (>0.62). Therefore, these are the most variable morphological characters among the five studied provinces. The PCA biplot, (not shown) revealed that morphological characters 3 and 10 differentiate mainly province 2 (Razavi Khorasan), while character 26 differentiates province 5 (Ardebil) from the others. ISSR analysis ISSR analysis of the studied provinces produced 97 reproducible bands. The PCO plot (Fig. 3) revealed that plants from different provinces were grouped together due to genetic similarity, for example those from prov- inces 2, 3 and 4. Therefore, ISSR data do not differenti- ate the studied provinces. This is in agreement with our morphometric analyses. Table 4. The mean value and standard error of quantitative morphological characters. Character Qazvin Razavi Khorasan Mazandaran Tehran Ardebil 14 specimens 14 specimens 21 specimens 11 specimens 9 specimens Leaf length of stem(mm) 15.50 ±0.73 8.00 ±0.49 12.30 ±1.16 15.18 ±0.74 10.07 ±0.36 Leaf width of stem(mm) 4.50 ±2.00 3.42 ±0.27 3.60 ±0.23 3.80 ±0.35 4.00 ±0.44 Stem Leaf length / width ratio 3.48 ±0.16 2.41 ±0.13 3.32 ±0.12 4.18 ±0.28 2.90 ±0.26 Petiole length(mm) 1.75 ±0.23 2.85 ±0.77 1.60 ±0.13 1.40 ±0.13 1.73 ±0.87 Inflorescence leaf length (mm) 7.04 ±1.03 4.94 ±0.44 6.74 ±0.62 6.77 ±0.84 7.35 ±0.63 Inflorescence leaf width (mm) 2.40 ±0.20 2.50 ±0.20 3.00 ±0.22 2.52 ±0.31 3.25 ±0.27 Inflorescence leaf length/ width ratio leaf width 3.31 ±0.31 2.50 ±0.11 2.23 ±0.11 2.80±0.21 2.30 ±0.31 Pedicle length (mm) 1.33 ±0.13 0.49 ±0.10 1.40 ±0.07 1.38 ±0.06 1.51 ±0.07 Calyx length(mm) 4.07 ±0.13 4.58 ±0.27 5.24 ±0.15 8.25 ±3.48 5.17 ±0.18 Calyx width(mm) 1.05 ±0.08 1.15 ±0.10 1.20 ±0.05 1.25 ±0.08 1.25 ±0.02 Calyx length/ width ratio 4.08 ±0.30 4.29 ±0.22 4.41 ±0.15 3.83 ±0.17 4.11 ±0.13 Calyx teeth length(mm) 0.78 ±0.07 0.96 ±0.06 0.93 ±0.06 0.81 ±0.09 0.82 ±0.05 Inflorescence length(cm) 1.63 ±0.06 1.33 ±0.09 1.37 ±0.08 1.29 ±0.13 1.45 ±0.17 Inflorescence width(cm) 1.84 ±0.05 1.82 ±0.06 1.62 ±0.07 1.59 ±0.09 1.53 ±0.14 Inflorescence length/ width ratio 0.86 ±0.03 0.82 ±0.05 0.83 ±0.03 0.83 ±0.08 0.94 ±0.05 Corolla length(mm) 5.87 ±0.27 6.21 ±0.33 6.28 ±0.27 6.12 ±0.26 6.21 ±0.45 Corolla tube length(mm) 3.48 ±0.16 3.57 ±0.19 3.80 ±0.18 3.43 ±0.17 4.03 ±0.38 Petal length(mm) 2.25 ±0.17 2.60 ±0.20 2.48 ±0.12 2.69 ±0.12 2.18 ±0.09 Corolla tube length/Petal length 1.54 ±0.10 1.48 ±0.13 1.56 ±0.08 1.26 ±0.06 1.84 ±0.13 Stamen length(mm) 1.19 ±0.16 2.07 ±0.28 1.78 ±0.22 0.68 ±0.21 1.99 ±0.26 Style length(mm) 4.78 ±0.23 4.81 ±0.41 5.13 ±0.28 4.59 ±0.28 4.54 ±0.29 Mean ± standard error. 105Population genetic and phylogeographic analyses of Ziziphora clinopodioides Moreover, the consensus tree of morphological and genetic features did not differentiate the plants collected in the studied provinces (Fig. not given), only distin- guishing plant numbers 6 and 7 of Qazvin province (Province 1), and plants 46 and 47 of Mazandaran prov- ince (Province 3). This result suggests that morphologi- cal variation in the studied provinces is not in agreement with their genetic features. Therefore, the present study does not support the idea that Z. clinopodioides contains any subspecies in Iran. This conclusion is further sup- ported by haplotype networking of cp-DNA (Fig. 4). The studied plants differed in cp-DNA sequences. The haplotype network separated outgroups from the studied Ziziphora clinopodioides plants. Moreover, it revealed large-scale within-province cp-DNA variation. For example, plants studied in Mazandaran, Ardebil and Razavi-Khorasan provinces were widely scattered on the network. Provincial genetic diversity analyses Genetic diversity parameters from the studied prov- inces are presented in Table 5. The highest value of genetic polymorphism in province 3 (79.38%) and the highest value of Nei gene diversity occurred in province 2 (0.158), while the lowest value of the same parameters was observed in province 5 (45.36% and 0.123, respec- tively). This indicates that province 5 has a lower degree of within province genetic variability. AMOVA and Gst results revealed significant differ- ence among the studied provinces. AMOVA produced a PhiPT value of 0.068 (P = 0.01), while the Gst value was 0.065 (P = 0.01). Pair-wise analysis of Fst and Gst revealed significant difference between provinces (Table 6). AMOVA revealed that 93.2% of total genetic vari- ability occurred due to within province diversity and 6.87% due to among province diversity. This is in agree- ment with PCO plot of ISSR data presented before; the provinces were not differentiated. Migration analysis of genetic data in all populations of five provinces produced a mean Nm value of 6.45 and Fig 4. Haplotype network of cp-DNA data in the studied Ziziphora clinopodioides provinces. Figure 2. PCA plot of Ziziphora clinopodioides provinces based on 29 morphological characters. Figure 3. PCO plot of Ziziphora clinopodioides provinces based on ISSR data. Table 5. Genetic diversity parameters in the studied provinces based on ISSR data Province N Na Ne I He UHe %P Hs Qazvin 14 1.340 1.191 0.227 0.134 0.139 67.01 0.218 Razavi Khorasan 14 1.464 1.227 0.262 0.158 0.163 73.20 0.251 Mazandaran 21 1.588 1.193 0.246 0.142 0.145 79.38 0.234 Tehran 11 1.361 1.197 0.242 0.143 0.149 68.04 0.242 Ardebil 9 0.907 1.192 0.195 0.123 0.130 45.36 0.185 N = No. plants, Na = No. alleles, Ne = No. effective alleles, I = Sha- non Information Index, He = Nei gene diversity, UHe = Unbiased gene diversity, %P = Per- centage of genetic polymorphism, and Hs = Genetic diversity due to population. 106 Raheleh Tabaripour et al. a Gst value of 0.07. These values indicate a high degree of gene flow among the studied populations. Moreover, STRUCTURE analysis based on a genetic admixture model also revealed a high degree of genetic admixture among the studied provinces as they had very similar allele combinations (similarly colored segments). These common shared alleles are either ancestral shared alleles or occurred due to ongoing gene flow among the popu- lations. The Evanno test identified two gene pools. The province assignment test revealed that gene flow occurred between all provinces but was higher between plants in provinces 1, 3 and 4. Province 5 had the low- est degree of gene flow as revealed by the lowest within province genetic variability as stated above. This prov- ince had limited gene flow with provinces 3 and 4. The pseudo-F value of K-Means clustering and Evanno test of STRUCTUR E revealed two genetic groups. When we performed the STRUCTURE analy- sis for k = 2 (Fig. 5), it revealed that provinces 1 and 5 formed the first genetic group, while provinces 2-4 com- prised the second genetic group. Therefore, we have two gene pools in Iran for this medicinal plant that can be used in germplasm conservation and future medicinal evaluation. The Mantel test produced significant correlation (r = 0.184, P = 0.01) between geographical distance and genetic distance of the studied provinces. This means that IBD (Isolation by distance) has occurred in Z. clino- podioides provinces and the neighboring provinces can exchange genes more frequently compared to those that are further from each other. This could be the reason for the higher degree of genetic similarity observed between provinces 2, 3 and 4. Divergence time estimation cp-DNA haplotypes can be considered as good molecular markers for investigating probable dates of appearance of populations and their paths of distribu- tion in the country (Minaeifar et al, 2016). BEAST and RASP analyses (Figs 6, 7) suggested that the oldest cp- DNA haplotype of Z. clinopodioides appeared sometime around 5-10 Mya in Mazandaran province (province 3). This suggests that Z. clinopodioides could possibly have appeared in the northern regions of the country during the Miocene era, with plants subsequently dispersing towards the north-eastern (Razavi Khorasan), north- western (Ardebil) and central parts of Iran (Tehran and Qazvin). DISCUSSION In the present study molecular markers such mul- tilocus ISSRs and cp-DNA sequences and morphologi- cal variables were used for genetic diversity, species and subspecies delimitation of Z. clinopodioides. According Table 6. Pair-wise analysis of Fst in the studied provinces based on ISSR data. Qazvin Razavi Khorasan Mazanda- ran Tehran Ardebil Qazvin 0.000 Razavi Khorasan 0.075 0.000 Mazandaran 0.035 0.030 0.000 Tehran 0.057 0.054 0.028 0.000 Ardebil 0.099 0.156 0.123 0.132 0.000 Figure 5. STRUCTURE plot of Ziziphora clinopodioides provinces based on k = 2. (Provinces 1-5 are: 1- Qazvin, 2- Razavi Khorasan, 3- Mazandaran, 4- Tehran, and 5- Ardebil). Figure 6. Chronogram from BEAST analysis of the studied prov- inces for Ziziphora clinopodioides based on the cp-DNA dataset (rpL16), showing 95% highest posterior density bars (HPD) in pur- ple. Numbers on nodes are clade credibility values. (Provinces 1-5 are: 1- Qazvin, 2- Razavi Khorasan, 3- Mazandaran, 4- Tehran, and 5- Ardebil). 107Population genetic and phylogeographic analyses of Ziziphora clinopodioides to several evaluations, phylogenetic markers (ITS and cpDNA) and ISSR molecular techniques are useful for genetic diversity, species and subspecies delimitation for different taxa such as, Diospyros L. (Li et al. 2018), Marrubium L. (Salehi et al. 2018), Carum L. (Papini et al. 2015), Acer velutinum Boiss (Siahkolaee 2017), Cycas diannanensis Z. T. Guan & G. D. Tao (Jian et al. 2015) and Petunia axillaris (Lam.) Britton, Sterns & Poggenb (Turchetto et al. 2014). Both molecular markers (multilocus ISSRs as well as cp-DNA sequences) and morphological characters pro- duced similar results and showed a lack of province dis- continuity within Z. clinopodioides. Therefore, our data do not suggest the presence of subspecies in the studied populations of this species. Jamzad (2012) in the Flora of Iran, after thorough morphological investigation in Z. cli- nopodioides, suggested that due to a high degree of mor- phological variability and co-occurrence of many sub- species in one location, she could not be sure about the number of subspecies within Z. clinopodioides and sug- gested the use of molecular studies to solve this problem. Moreover, hig h mor pholog ica l, pa ly nolog ica l and molecular diversity exist among Ziziphora taxa (Tabaripour et al. 2018; Tabaripour et al. 2019) and the genus shows very variable chromosome number along a descending dysploidy line starting from 2n = 16 to 2n = 34 (Taarna 1973; Selvi et al. 2013), but Z. clinopodioides proved to has 2n=18 (Selvi et al. 2013). In a similar study, subspecies determination was con- ducted in the Western Australian species Pityrodia scabra A.S. George. (Lamiaceae) using a combined approach with non-coding chloroplast gene regions and morpho- logical data (Shepherd et al. 2013). They observed that some morphological features varied among the popu- lations and provided some evidence for cryptic taxa. Furthermore, molecular phylogenetic analyses revealed genetic distinctiveness between the Wyalkatchem (type) population and the Southern Cross and Lake Lefroy pop- ulations. This evidence, when used in conjunction with the morphological differences, provided support for the recognition of the new subspecies described as Pityrodia scabra subsp. dendrotricha K.A. Sheph. subsp. nov. Population genetics studies are an important step in planning genetic and breeding programs for crop and medicinal plants. They provide data on genetic variabili- ty, gene flow versus population genetic isolation, popula- tion genetic fragmentation, alongside the role of genetic drift, bottlenecks and other evolutionary forces acting on population divergence (Sheidai et al. 2013, 2014). With increases in sizes of human populations, crop plants and medicinally important plant taxa are con- sumed and destroyed faster than before. Medicinal plants such as Z. clinopodioides are extensively used by locals and therefore potentially threatened in their natu- ral habitats. Therefore, to design an effective conserva- tion strategy, knowledge of genetic diversity in the target species is important. The present study revealed a high level of morpho- logical and genetic variability both within and among provinces of Z. clinopodioides. AMOVA revealed that 93% of total genetic variability occurred due to within province diversity and 7% due to among province diver- sity. This could be due to the out-crossing nature of this species. These plants are usually cross pollinated in nature by insects, which can result in high within popu- lation genetic variability. We can exploit this variability in future hybridization and breeding strategies. Assessments of levels of within- and among-popula- tion genetic variations have been used to prioritize pop- ulations for conservation efforts (Petit et al. 1998), with (all else being equal) more weight given to populations exhibiting higher levels of within-population variation and to those that are more genetically divergent. The STRUCTURE plot and province assignment revealed some degree of genetic admixture among the Fig 7. Cp-DNA RASP analysis based on BEAST tree (MCMC) showing probable ancestral area distribution for Ziziphora clinopo- dioides provinces. Razavi Khorasan, B- Ardebil, C- Mazandaran, D- Qazvin, E- Tehran and F- Lellemanthia (outgroup). 108 Raheleh Tabaripour et al. studied Z. clinopodioides provinces. Gene flow is also important in conservation contexts, particularly for species with local populations. Fortunately, Z. clinopo- dioides provinces showed high within-province genetic variability and high among province gene flow. Gene flow among local populations could mitigate losses of genetic variation caused by genetic drift in local popula- tions and potentially save them from extinction (Sheidai et al. 2014; Safaei et al. 2016). The Mantel test revealed isolation by distance in the studied Z. clinopodioides provinces. In plant species that form geographical populations, as geographical isolation increases, a reduction in both seed dispersal and pollen flow will result in decreased gene flow between distantly located populations (Freeland et al. 2011). This explains why the Evanno test and K-Means clustering identified two different gene pools for Z. clinopodioides within the country. BEAST and RASP results suggested that Z. clino- podioides haplotypes appeared around 5-10 Mya in the mountainous regions of Qazvin and Mazandaran. Active divergence occurred between 1-5 Mya in these mountains due to their reactions to Pleistocene glacia- tions. 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