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Molecular investigation in few spices of Dacocephalum in Iran: Species relationship, reticulation and divergence time
Industrial Crops & Products 141 (2019) 111758
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Molecular investigation in few spices of Dacocephalum in Iran: Species relationship, reticulation and divergence time
T
Fahimeh Koohdar , Masoud Sheidai ⁎
Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran
ARTICLE INFO
ABSTRACT
Keywords: Dracocephalum cp-DNA Molecular phylogeny BEAST Hybridization
It is believed that Lamiaceae or the Mint family has numerous paraphyletic genera and the relationship between the species has not been fully determined in several cases. The genus Dracocephalum is one of the genera, which has not been investigated thoroughly. In the family Lamiaceae, Dracocephalum is the second largest genus consisting of about 186 species. Eight Dracocephalum species were reported of Iran belonging to the IranoTuranian phytogeographical region. The present study has attempted to investigate Dracocephalum in terms of the molecular phylogeny for the first time and gather data on their species relationship, divergence time, and show how they are distributed geographically. To report the results, Chloroplast DNA (ribosomal protein L16 (rpL16)) sequences and Inter Simple Sequence Repeat (ISSR) molecular markers have been used. According to the results, the genus Dracocephalum is paraphyletic and has close affinity with Lallemantia. The results of horizontal gene transfer (HGT) analysis confirmed the inter-specific hybridization in the genus. Chronogram obtained by Bayesian Evolutionary Analysis Sampling Trees (BEAST) date revealed Dracocephalum species divergence time back to 2–10 million years ago(MYA). These findings extend the knowledge on the evolution of Lamiaceae.
1. Introduction
have been discovered as derivatives of alkaloids, flavonoids, terpenoids, and others from Dracocephalum species (Zeng et al., 2010). For instance, D. moldavica L. has been used in the ethno medicine of Europe to cure hypertension and heart disease (Dastmalchi et al., 2007; Olennikov et al., 2013). Besides, in Xinjiang to cure asthma and gastropathy, D. heterophyllum Benth. was used. Moreover, D. nutans L. was used to treat stomach-related diseases and liver in Tibet (Singh, 2012). Badrandjboie-Dennaie and Zarrin-Giah (D. kotschyi Boiss. in Iran), is favoured as an important medicinal herbaceous plant (Fattahi et al., 2011). D. kotschyi have used as sources rich in valuable essential oils and flavonoids by Locals (Sajjadi et al., 1998). It is believed that its seeds provide a large supply of linoleic, oleic and linolenic acids (Goli et al., 2012). Sometimes, The Persian name of Melissa officinalis (badranjboye) resemble D. kotschyi in Iran market. According to Flora Iranica, there are eight Dracocephalum species (Rechinger, 1982), but Jamzad (2012) believed there are 10 species in Flora of Iran, out of which five are native to Iran. Using Dracocephalum species by human beings has a long history, but scientific data on the genus is limited and sufficient data is not available on their phylogeny, molecular systematic and evolution (Sonboli et al., 2011; Drew and Sytsma, 2012; Salehi et al., 2014). According to the Karyological study on two Dracocephalum species of
The genus Dracocephalum L., which is also called dragonhead (Lamiaceae), has about 186 species (Budantsev, 1987; Sonboli et al., 2011). Dracocephalum species are typically perennial herbs growing in alpine and semi-dry regions. They grow mostly in moderate Asian regions (Brach and Song, 2006); Verticillasters in this genus subtended by floral leaves or bracts. This genus produces inflorescences that are a lax oblong or compact globose. The flowers usually have bracteoles entire or 3-fid. Calyx is tubular or tubular-campanulate with 15-vein and 2- lip that upper lip has 3-dentate and lower lip has 2-dentate. Corolla is 2lipped with tube narrow and without longitudinal folds. Flowers have four stamens with glabrous anthers. Style has 2 equal lobes. Nutlets are oblong and smooth (Davis, 1982). This genus is closely related to Lallemantia but it differs from Lallemantia in having the upper lip of corolla without folds and distinctively veins bracteoles which are aristate (Edmondson, 1982). It is believed that Dracocephalum species have been used in anticancer treatments, analgesic, antioxidant and antimicrobial (Sajjadi et al., 1998; Zeng et al., 2010; Sonboli et al., 2011). Ethno-pharmacological information shows that Dracocephalum species have been used for medications. Around a hundred compounds ⁎
Corresponding author. E-mail addresses: [email protected] (F. Koohdar), [email protected] (M. Sheidai).
https://doi.org/10.1016/j.indcrop.2019.111758 Received 6 January 2019; Received in revised form 2 September 2019; Accepted 3 September 2019 Available online 10 September 2019 0926-6690/ © 2019 Published by Elsevier B.V.
Industrial Crops & Products 141 (2019) 111758
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2. Material and methods
using the following procedure: 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, followed by one final extension at 72 °C for 7 min. To visualize the result of amplification, it was run on 2% agarose gel, followed by the ethidium bromide staining. A 100 base pair (bp) molecular size ladder (Fermentas, Germany) was used to determine the fragment size. As specified by Shaw and Small (2005) it was tried to amplify the intron in the gene for ribosomal protein L16 (rpL16) located in the chloroplast genome and sequenced with 0.2 μM primer. A 25 μl volume containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany), 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany) was used to carry out PCR reactions. The following thermocycler parameters were used: 94 °C for 5 min, followed by 35 cycles at 98 °C for 10 s, 50–55 °C for 40 s, and 72 °C for 2 min, followed by one final extension at 72 °C for 7 min. To visualize PCR products, 2.5% agarose gel through GelRed™ Nucleic Acid Gel Staining was applied. A 100 bp size ladder was used to determine the fragment size.
2.1. Plant materials
2.3. Data analyses
In this study, for ISSR marker73 plant specimens of 7 Dracocephalum species and 5 Lallemantia Fisch. & C.A.Mey. species (out group) were randomly collected. One sample of any species (7 Dracocephalum species, 5 species Lallemantia (out group) and2 species of ziziphora L. (out group)) for cp-DNA marker were used. The voucher specimens were deposited in Herbarium of Shahid Beheshti University (HSBU) (Table 1).
2.3.1. ISSR analyses Binary characters (presence = 1, absence = 0) were used to encode ISSR bands. The grouping of the species was done by Minimum spherical cluster method (WARD) (Podani, 2000). Paleontological statistics (PAST) ver. 2.17 (Hammer et al., 2012) was used for analysis. For ISSR marker in populations of D. kotschyi, D. multicaule Montbret & Aucher ex Benth., D. thymiflorum L. and D. aucheri Boiss, genetic diversity parameters such as the percentage of allelic polymorphism, allele diversity, Nei’s gene diversity (He), and Shannon information index (I) were specified (Weising et al., 2005). GenAlEx 6.4 was used for these analyses (Peakall and Smouse, 2006). We performed K-Means clustering as done in GenoDive ver. 2. (2013). Clustering having the smallest amount of variation within clusters was considered optimum, which was determined using the within-clusters sum of squares. The minimization of the within-groups sum of squares used in K-Means clustering is, in the context of a hierarchical Analysis of molecular variance (AMOVA), equivalent to minimizing the among-populations-within-groups sum of squares, SSDAP/ WG (Meirmans, 2012). The best fit for k (Meirmans, 2012) is obtained by two summary statistics, 1- pseudo-F (Calinski and Harabasz, 1974), and 2- Bayesian Information Criterion (BIC).
Iran, D. moldavica is diploid with x = 5 (2n = 2x = 10) and D. kotschyi is tetraploid (Salehi et al., 2014). In general, limited literature exists on molecular details of the genus Dracocephalum yet and no detailed information is available on its pattern of speciation, reticulation and the species divergence time. For example, Koohdar et al. (2015) and Sheidai and Koohdar (2018) reported the probability of two varieties in Dracocephalum thymiflorum based on ISSR, morphology and anatomy studies. Similarly, Koohdar et al. (2018) reported the probability of hybrid between Dracocephalum kotschyi and Dracocephalum oligadenium based on evidence morphological, anatomical and molecular data. Therefore, the aims of present study are to illustrate the species phylogenetic relationship, and provide data on their divergence time and path of geographical distribution. This information can add up to the knowledge on Lamiaceae evolution. In general, limited literature exists on the biosystematics and molecular studies on the genus Dracocephalum.
2.2. DNA extraction, amplification and sequencing Fresh leaves were put to dry in silica gel powder. Cetyltrimethylammonium bromide -activated charcoal protocol (CTAB) was applied to extract the genomic DNA. The extraction was done by activating charcoal and poly vinyl pyrrolidone (PVP) for binding of polyphenolics during extraction; for mild extraction and precipitation conditions, the high-molecular weight DNA isolation was boosted without the interference of impurities. The extracted DNA was examined in terms of quality and quantity by running on 0.8% agarose (Sheidai et al., 2013). Eight ISSR primers; (CA) 7 G T, (AGC) 5 G G, UBC 810, (GA) 9C, UBC 807, UBC 811, (GT) 7CA and (GA) 9A commercialized by UBC (the University of British Columbia) were used (Sheidai et al., 2012). Using a 25 μL volume containing 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany); 50 mM KCl; 10 mM Tris-HCl buffer at pH;8 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of each primer, polymerase chain reaction (PCR) was implemented. The reactions were amplified in Techne thermocycler (Germany)
2.3.2. Cp-DNA sequences analyses To investigate the species relationship like Networking and Bayesian approach, several phylogenetic methods were applied. MUSCLE program was applied to align the intron in the gene for ribosomal protein L16 (rpL16) first; then, it was used to examine the
Table 1 Dracocephalum, Lallemantia and Ziziphora species studied their localities and voucher numbers. No.
Province
Locality
Voucher number
Number of samples for ISSR study
1 2 3 4 5 6 7 8 9 10 11 12 13 14
D. kotschyi D. thymiflorum D. multicaule D. aucheri D. moldavica D. subcapitatum D. lindbergii L. peltata L. canescens L. baldschuanica L. iberica L. royleana Z. tenuior Z. persica
Mazandaran Mazandaran Qazvin Tehran Orumiyeh Khorasan Khorasan Alborz Qazvin Khorasan Zanjan Khorasan Qazvin Qazvin
HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU
7 6 5 9 5 3 5 9 5 6 5 8 – –
201213 201214 201215 201216 201217 201218 201219 201209 201200 201208 201210 201212 201220 201221
2
MH568733 MH453498 MH453499 MH453500 MH453497
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appropriate nucleotide substitution model as carried out in molecular evolutionary genetics analysis (MEGA) 7. Program (Tamura et al., 2012). Splits Tree4 program and BEAST software v1.6.1 (Drummond et al., 2010a, b) were used for networking and Bayesian analysis, respectively. 2.3.3. Time of species divergence To estimate divergence time in the studied species, Chloroplast DNA sequences were applied and for performing analyses, BEAST v1.6.1 was used (Drummond et al., 2010a, b). To have the initial xml files for BEAST, Bayesian evolutionary analysis utility version (BEAUti) v1.6.1 was used (Drummond et al., 2010a, b). The Yule tree prior is broadly known for providing the best-fit model for trees defining the relationships between various species that is capably of describing the net speciation rate (Drummond et al., 2010a, 2010b; Nee, 2006). For the MCMC analyses, the chain length was 10000000. 10000 trees were put for the analyses following the removal of 100 trees representing the burn-in. The BEAUti xml file was run in BEAST v1.6.1 (Drummond et al., 2010a, 2010b). Since there was were not any fossils for the target species, a rate of evolution of the plastid sequence (u = 1.0 × 10 −9 s s-1 year-1; Zurawski et al., 1984; Minaeifar et al., 2016) was considered that was entered in the option of molecular clock model in BEAUti v1.6.1. The normal distribution (Mean = 0, St. dev = 1) was used for priors. To test the sampling and convergence, Tracer v.1.5 was applied (Drummond and Rambaut, 2007). To explain the phylogenetic results generated by BEAST to form a single ‘target’ tree (maximum clade credibility tree (MCC)) including summary statistics, Tree Annotator v1.6.1 was applied. To generate the annotated BEAST (MCC) tree, FigTree v1.3.1 (Rambaut, 2009) was used. To analyze the biogeography, Cp-DNA data of the present study was used.
Fig. 1. ISSR profiles of 13 accessions of Dracocephalum using the primer UBC811. L: represent molecular weight size marker (100 kb ladder). N: negative control. The numbers represent different accessions according to Table 1.
polymorphism (44.23%) among the studied Dracocephalum species (Table 3). It has a wide geographical distribution in the country and occurs in Northern Provinces of Gorgan, Mazandaran, and Gilan, as well as in the Northeast Province of Azarbayejan and central Iran (Semnan and Tehran Provinces). Two species of D. thymiflorum and D. aucheri had almost similar level of genetic polymorphism. D. thymiflorum occurs in Mazandaran, Gilan, and Azarbayejan Provinces, and D. aucheri grows in Mazandaran, Tehran, Semnan, and Ghazvin. Dracocephalum multicuale had the lowest degree of genetic variability. It has also a confined geographical distribution and occurs only in two provinces of Ghazvin and Azarbayejan.
2.3.4. Biogeography The Bio- and phylogeographic markers are specially DNA sequences, yet, recently fingerprinting techniques, like simple sequence repeats (SSRs), amplified fragment length polymorphism (AFLPs), randomly amplified polymorphic DNA (RAPDs), and ISSRs, are also often used for phylogeographic investigations, because of the generally high levels of variability retrieved (Clausing et al., 2000; Hess et al., 2000; Pleines et al., 2009). To analyze biogeography, Reconstruction Ancestral State in Phylogenies program (RASP) programs was used (Yu et al., 2015). To infer ancestral state using statistical dispersal-vicariance analysis (SDIVA), RASP is usually applied. In the present study, both binary markov chain monte carlo (MCMC) and S- DIVA methods of RASP were applied for analyses. And based on ancestral of species distribution, the route of these species in the country was reconstructed.
3.2. Monophyly versus paraphyly in Dracocephalum WARD diagram presented previously based on ISSR data revealed that out-group species of Lallemantia are scattered among Dracocephalum species (Fig. 2). This indicated close affinity between these two genera and that they are not monophyletic. Close relationship between the species in both genera was supported by pair-wise AMOVA (Table 4) and the results did not show significant molecular difference between these species (FST = 0.17, P = 0.01). Therefore, although we obtained well delimited species based on K-Means clustering, they do not show significant molecular difference. However, when we compared the pooled data in each genus, AMOVA produced significant molecular difference; this indicates some degree of genetic differentiation between Dracocephalum and Lallemantia. This is further supported by K-Means clustering of both genera as the test produced optimal k = 2. However, in a phylogenetic context, these two genera are paraphyletic as evidenced by WARD analysis (Fig. 2). The representative species of Lallemantia were scattered among Dracocephalum species. Phylogenetic relationships of the studied species are provided in Bayesian tree of BEAST and TCS network (Figs. 3 and 4). The out-group species of Ziziphora formed a separate clade while the other species in Dracocephalum and Lallemantia were placed intermixed to some extent. Dracocephalum subcapitatum (Kuntze) Lipsky, D. kotschyi and D.
3. Results 3.1. Species delimitation and genetic diversity analyses by ISSR markers Out of a total of 8 different primers screened, only 6 primers ((CA) 7 G T, UBC 810, (GA) 9C, UBC 811, (GT) 7CA and (GA) 9A) produced reproducible and polymorphic bands for all species representing the taxa studied (Fig. 1). Clustering and network analyses of the studied species from Dracocephalum and Lallemantia (out-group taxa) produced similar groupings; therefore, only WARD is presented here (Fig. 2). The specimens of Dracocephalum species were almost delimited in a single cluster or group. Moreover, K-Means (Table 2) clustering recognized all 9 species (D. kotschyi, D. multicaule, D. thymiflorum, D. aucheri, and 5 species of Lallemantia) based on ISSR data, therefore the species rank is confirmed and they are delimited based on ISSR molecular markers. Dracocephalum kotschyi showed the highest degree of genetic 3
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Fig. 2. WARD diagram in Dracocephalum species based on cp-DNA. Table 2 K-Means clustering of the studied species.
Table 4 Paired-sample AMOVA between Dracocephalum and Lallemantia species.
k
SSD(T)
SSD(AC)
SSD(WC)
r-squared
pseudo-F
AIC
1 2 3 4 5 6 7 8 9&*
274.423 274.423 274.423 274.423 274.423 274.423 274.423 274.423 274.423
0.000 37.525 68.123 96.68 119.663 139.368 157.034 174.590 189.923
0.000 237.371 206.300 178.356 154.760 135.056 117.389 99.833 84.500
0.000 0.135 0.248 0.350 0.436 0.508 0.572 0.636 0.692
0.000 3.746 3.797 3.950 4.059 4.128 4.263 4.497 4.776
63.438 62.022 60.943 59.973 59.378 59.258 59.419 59.451 59.895
Pop1 Pop2 Pop3 Pop4 Pop5 Pop6 Pop7 Pop8 Pop9 a
* Best clustering according to Calinski & Harabasz' pseudo-F: k = 9. & Best clustering according to Bayesian Information Criterion: k = 9.
N
Na
Ne
I
He
uHe
%P
D. D. D. D.
10 10 10 7
0.885 0.683 0.467 0.673
1.160 1.175 1.098 1.192
00.174 0.164 0.090 0.168
0.107 0.107 0.058 0.111
0.113 0.113 0.061 0.120
44.23 33.65 22.12 32.69
kotschy thymiflorum multicuale aucheri
Pop2
Pop3
Pop4
Pop5
Pop5
Pop7
Pop8
Pop9
– 0.096 0.121 0.085 0.082 0.117 0.109 0.094 0.096
0.306 – 0.114 0.091 0.092 0.092 0.107 0.096 0.103
0.416 0.420 – 0.095 0.106 0.100 0.094 0.102 0.094
0.320 0.537 0.554 – 0.113 0.101 0.108 0.092 0.097
0.494 0.631 0.633 0.781 – 0.110 0.091 0.110 0.102
0.222 0.456 0.440 0.393 0.511 – 0.110 0.105 0.123
0.405 0.546 0.548 0.650 0.656 0.420 – 0.095 0.120
0.522 0.582 0.655 0.752 0.882 0.497 0.671 – 0.091
0.531 0.722 0.753 0.804 0941 0.643 0.652 0.936 –
Above diagonal : FST value, bellow diagonal : P value.
aucheri were placed close to each other. These species along with Lallemantia baldschuanica Gontsch., L. iberica (M.Bieb.) Fisch. & C.A.Mey., L. canescens (L.) Fisch. & C.A.Mey., and D. moldavica formed the second major clade. Dracocephalum multicuale and D. thymiflorum showed close affinity and comprised a single clade. They joined the afore-mentioned clade with some distance. Lallemantia peltata (L.) Fisch. & C.A.Mey., L. royleana (Benth.) Benth., and D. lindbergii Rech.f., showed affinity and formed the forth clade, separated from the other studied species. The method of templeton (TCS) haplotype network supported BEAST phylogenetic relationship, and revealed the presence of three main groups (Figs. 3 and 4). The first group was formed by D. subcapitatum, D. kotschyi, D. multicuale and D. thymiflorum. The second haplotype group was composed of L. peltata, L. royleana, and D.
Table 3 Genetic diversity parameters in the studied Dracocephalum species. Species
Pop1
a
N: No of plants studied. Na: No. of alleles. c Ne : Effective No. of alleles. d He: Gene diversity. e UHe : Unbiased gene diversity. f %P: Polymorphism percentage. b
4
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Fig. 3. BEAST chronogram of the studied species based on cp-DNA.
Dracocephalum occurred between 2–10 MYA. RASP analysis revealed that the most probable ancestral area of Dracocephalum species distribution in Iran is Mazandaran province located in the northern Iran. Two species of D. kotschyi and D. thymiflorum occurred in this province (No figure). Ghazvin province is the second probable ancestral area of Dracocephalum and it is occupied by D. multicuale. The more recent areas of the country occupied by Dracocephalum species are Tehran and Khorasan provinces. 3.4. HGT analysis The reticulogram produced by T-REX program showed horizontal gene transfer (HGT) between D. moldavica and D. subcapitalum, and also between D. moldavica and both L. iberica and L. canescens (Fig. 5). The HGT obtained is in agreement with inter-mixed positions obtained for the species in these two genera in both ISSR and Cp-DNA trees. HGT is the probable reason for the occurrence of common shared alleles in
Fig. 4. TCS haplotype network of cp-DNA.
lindbergii; while the other studied species formed the third major haplotype group. Further evidence in support of close genetic relationship between species in Dracocephalum and Lallemantia was obtained by AMOVA of Cp-DNA sequences. When AMOVA was performed among members of three genera viz. Dracocephalum, with those of out-groups Lallemantia and Ziziphora, it produced no significant molecular difference (Phi pt = 0.01, P .0.05). Nevertheless, when it was performed between combined Dracocephalum + Lallemantia with species in Ziziphora, a significant molecular difference was obtained (Phi pt = 0.17, **P < 0.01). 3.3. Time of divergence and ancestral area The chronogram of BEAST (Fig. 3) suggests that Ziziphora, Lallemantia and Dracocephalum studied species become diverged 30–80, while the more recent species divergence within Lallemantia and
Fig. 5. HGT tree showing gene flow among the studied Dracocephalum. 5
Industrial Crops & Products 141 (2019) 111758
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Declaration of Competing Interest
these two genera, which in turn brings about their genetic similarity.
The authors declare no conflicts of interest.
4. Discussion
Acknowledgment
The sixth largest family of flowering plants with economic value is Lamiaceae family. Seven subfamilies were determined by Harley et al. (2004) within Lamiaceae. This classification influenced largely by morphological features and recent molecular reports (Drew and Sytsma, 2012). Apparently, Lamiaceae has numerous paraphyletic and genera that have not been investigated; for instance, Bendiksby et al. (2011) suggested the transfer of species to put the monophyly in genus Lamium L. and Otostegia Benth.. They reported that numerous genera are nonmonophyletic including Ballota s.str., Lagopsis (Bunge ex Benth.) Bunge, Leonotis (Pers.) R.Br., Leonurus L., Leucas R.Br., Microtoena Prain., Phlomoides Moench., Sideritis L., Stachys L., and Thuspeinanta T.Durand. Based on this study, Dracocephalum is a paraphyletic genus and this is consistent with the previous results (Drew and Sytsma, 2012). Lamiaceae genera developed along with extensive inter-specific hybridization and polyploidy events; thus understanding the species relationship will not be easy, as there is incongruence between phylogenetic trees from diverse sets of genes (Trusty et al., 2004; Salmaki et al., 2013). The present study highlighted the horizontal gene transfer (HGT) between D. moldavica and D. subcapitalum, and also between D. moldavica and both L. iberica and L. canescens. In our study we found that ISSRs can delimit Dracocephalum species. These dominant molecular markers are known to delimit different plant taxonomic groups like Salvia L., Cirsium Mill., Olive L., and Tamarix L., etc. (see for example, Sheidai et al., 2012, 2013; Sheidai et al., 2014; Ijbari et al., 2014; Safaei et al., 2016). Sonboli et al. (2011) studied Dracocephalum species and reported that RAPD markers can be used in species delimitation. The split between Phrymaceae + Linderniaceae and Lamiaceae, dates from 60 to 107 MYA (Drew and Sytsma, 2012). Lamiaceae are not well characterized in the fossil record (Harley et al., 2004), however Kar (1996) recognized the fossil Ocimum L., which is within Nepetoideae, and dates back to 49-MYA and the Early Eocene. In the EarlyMiddle Oligocene, A fossil fruit of Melissa was described that dates back to 28.4 MYA (Reid and Chandler, 1926; Martínez-Millán, 2010). Until now, Drew and Sytsma (2012) have used Cp-DNA to specify divergence time in Nepetoideaebased on fossil calibration. They believed that subfamily Nepetoideae extended around 57 Ma at the Paleocene-Eocene border. The development of the extant members of tribe Mentheae took place 46 MYA in the mid-Eocene; yet, all the evolution of the sub-tribes was by the Eocene-Oligocene border (ca. 34 MYA). All sub-tribes underwent generic differentiation in late Miocene or early Pliocene except Menthinae. Similarly, generic differentiation of Old World (primarily Mediterranean) Menthinae also happened during the late Miocene; yet, the North and South American sub-clade of Menthinae underwent differentiation far more recent in the Pliocene (Drew and Sytsma, 2012). The present study is in agreement with the findings of Drew and Sytsma (2012) and suggests that Ziziphora, Lallemantia and Dracocephalum studied species become diverged 30–80 mya, while the more recent species divergence within Lallemantia and Dracocephalum occurred between 2–10 MYA.
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5. Conclusion In conclusion, the present study revealed the species relationship within genus Dracocephalum and highlighted the potential role of interspecific hybridization in species diversification. This information extends the evolutionary knowledge of Lamiaceae in general. 6
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7
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Molecular investigation in few spices of Dacocephalum in Iran: Species relationship, reticulation and divergence time
T
Fahimeh Koohdar , Masoud Sheidai ⁎
Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran
ARTICLE INFO
ABSTRACT
Keywords: Dracocephalum cp-DNA Molecular phylogeny BEAST Hybridization
It is believed that Lamiaceae or the Mint family has numerous paraphyletic genera and the relationship between the species has not been fully determined in several cases. The genus Dracocephalum is one of the genera, which has not been investigated thoroughly. In the family Lamiaceae, Dracocephalum is the second largest genus consisting of about 186 species. Eight Dracocephalum species were reported of Iran belonging to the IranoTuranian phytogeographical region. The present study has attempted to investigate Dracocephalum in terms of the molecular phylogeny for the first time and gather data on their species relationship, divergence time, and show how they are distributed geographically. To report the results, Chloroplast DNA (ribosomal protein L16 (rpL16)) sequences and Inter Simple Sequence Repeat (ISSR) molecular markers have been used. According to the results, the genus Dracocephalum is paraphyletic and has close affinity with Lallemantia. The results of horizontal gene transfer (HGT) analysis confirmed the inter-specific hybridization in the genus. Chronogram obtained by Bayesian Evolutionary Analysis Sampling Trees (BEAST) date revealed Dracocephalum species divergence time back to 2–10 million years ago(MYA). These findings extend the knowledge on the evolution of Lamiaceae.
1. Introduction
have been discovered as derivatives of alkaloids, flavonoids, terpenoids, and others from Dracocephalum species (Zeng et al., 2010). For instance, D. moldavica L. has been used in the ethno medicine of Europe to cure hypertension and heart disease (Dastmalchi et al., 2007; Olennikov et al., 2013). Besides, in Xinjiang to cure asthma and gastropathy, D. heterophyllum Benth. was used. Moreover, D. nutans L. was used to treat stomach-related diseases and liver in Tibet (Singh, 2012). Badrandjboie-Dennaie and Zarrin-Giah (D. kotschyi Boiss. in Iran), is favoured as an important medicinal herbaceous plant (Fattahi et al., 2011). D. kotschyi have used as sources rich in valuable essential oils and flavonoids by Locals (Sajjadi et al., 1998). It is believed that its seeds provide a large supply of linoleic, oleic and linolenic acids (Goli et al., 2012). Sometimes, The Persian name of Melissa officinalis (badranjboye) resemble D. kotschyi in Iran market. According to Flora Iranica, there are eight Dracocephalum species (Rechinger, 1982), but Jamzad (2012) believed there are 10 species in Flora of Iran, out of which five are native to Iran. Using Dracocephalum species by human beings has a long history, but scientific data on the genus is limited and sufficient data is not available on their phylogeny, molecular systematic and evolution (Sonboli et al., 2011; Drew and Sytsma, 2012; Salehi et al., 2014). According to the Karyological study on two Dracocephalum species of
The genus Dracocephalum L., which is also called dragonhead (Lamiaceae), has about 186 species (Budantsev, 1987; Sonboli et al., 2011). Dracocephalum species are typically perennial herbs growing in alpine and semi-dry regions. They grow mostly in moderate Asian regions (Brach and Song, 2006); Verticillasters in this genus subtended by floral leaves or bracts. This genus produces inflorescences that are a lax oblong or compact globose. The flowers usually have bracteoles entire or 3-fid. Calyx is tubular or tubular-campanulate with 15-vein and 2- lip that upper lip has 3-dentate and lower lip has 2-dentate. Corolla is 2lipped with tube narrow and without longitudinal folds. Flowers have four stamens with glabrous anthers. Style has 2 equal lobes. Nutlets are oblong and smooth (Davis, 1982). This genus is closely related to Lallemantia but it differs from Lallemantia in having the upper lip of corolla without folds and distinctively veins bracteoles which are aristate (Edmondson, 1982). It is believed that Dracocephalum species have been used in anticancer treatments, analgesic, antioxidant and antimicrobial (Sajjadi et al., 1998; Zeng et al., 2010; Sonboli et al., 2011). Ethno-pharmacological information shows that Dracocephalum species have been used for medications. Around a hundred compounds ⁎
Corresponding author. E-mail addresses: [email protected] (F. Koohdar), [email protected] (M. Sheidai).
https://doi.org/10.1016/j.indcrop.2019.111758 Received 6 January 2019; Received in revised form 2 September 2019; Accepted 3 September 2019 Available online 10 September 2019 0926-6690/ © 2019 Published by Elsevier B.V.
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2. Material and methods
using the following procedure: 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, followed by one final extension at 72 °C for 7 min. To visualize the result of amplification, it was run on 2% agarose gel, followed by the ethidium bromide staining. A 100 base pair (bp) molecular size ladder (Fermentas, Germany) was used to determine the fragment size. As specified by Shaw and Small (2005) it was tried to amplify the intron in the gene for ribosomal protein L16 (rpL16) located in the chloroplast genome and sequenced with 0.2 μM primer. A 25 μl volume containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany), 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany) was used to carry out PCR reactions. The following thermocycler parameters were used: 94 °C for 5 min, followed by 35 cycles at 98 °C for 10 s, 50–55 °C for 40 s, and 72 °C for 2 min, followed by one final extension at 72 °C for 7 min. To visualize PCR products, 2.5% agarose gel through GelRed™ Nucleic Acid Gel Staining was applied. A 100 bp size ladder was used to determine the fragment size.
2.1. Plant materials
2.3. Data analyses
In this study, for ISSR marker73 plant specimens of 7 Dracocephalum species and 5 Lallemantia Fisch. & C.A.Mey. species (out group) were randomly collected. One sample of any species (7 Dracocephalum species, 5 species Lallemantia (out group) and2 species of ziziphora L. (out group)) for cp-DNA marker were used. The voucher specimens were deposited in Herbarium of Shahid Beheshti University (HSBU) (Table 1).
2.3.1. ISSR analyses Binary characters (presence = 1, absence = 0) were used to encode ISSR bands. The grouping of the species was done by Minimum spherical cluster method (WARD) (Podani, 2000). Paleontological statistics (PAST) ver. 2.17 (Hammer et al., 2012) was used for analysis. For ISSR marker in populations of D. kotschyi, D. multicaule Montbret & Aucher ex Benth., D. thymiflorum L. and D. aucheri Boiss, genetic diversity parameters such as the percentage of allelic polymorphism, allele diversity, Nei’s gene diversity (He), and Shannon information index (I) were specified (Weising et al., 2005). GenAlEx 6.4 was used for these analyses (Peakall and Smouse, 2006). We performed K-Means clustering as done in GenoDive ver. 2. (2013). Clustering having the smallest amount of variation within clusters was considered optimum, which was determined using the within-clusters sum of squares. The minimization of the within-groups sum of squares used in K-Means clustering is, in the context of a hierarchical Analysis of molecular variance (AMOVA), equivalent to minimizing the among-populations-within-groups sum of squares, SSDAP/ WG (Meirmans, 2012). The best fit for k (Meirmans, 2012) is obtained by two summary statistics, 1- pseudo-F (Calinski and Harabasz, 1974), and 2- Bayesian Information Criterion (BIC).
Iran, D. moldavica is diploid with x = 5 (2n = 2x = 10) and D. kotschyi is tetraploid (Salehi et al., 2014). In general, limited literature exists on molecular details of the genus Dracocephalum yet and no detailed information is available on its pattern of speciation, reticulation and the species divergence time. For example, Koohdar et al. (2015) and Sheidai and Koohdar (2018) reported the probability of two varieties in Dracocephalum thymiflorum based on ISSR, morphology and anatomy studies. Similarly, Koohdar et al. (2018) reported the probability of hybrid between Dracocephalum kotschyi and Dracocephalum oligadenium based on evidence morphological, anatomical and molecular data. Therefore, the aims of present study are to illustrate the species phylogenetic relationship, and provide data on their divergence time and path of geographical distribution. This information can add up to the knowledge on Lamiaceae evolution. In general, limited literature exists on the biosystematics and molecular studies on the genus Dracocephalum.
2.2. DNA extraction, amplification and sequencing Fresh leaves were put to dry in silica gel powder. Cetyltrimethylammonium bromide -activated charcoal protocol (CTAB) was applied to extract the genomic DNA. The extraction was done by activating charcoal and poly vinyl pyrrolidone (PVP) for binding of polyphenolics during extraction; for mild extraction and precipitation conditions, the high-molecular weight DNA isolation was boosted without the interference of impurities. The extracted DNA was examined in terms of quality and quantity by running on 0.8% agarose (Sheidai et al., 2013). Eight ISSR primers; (CA) 7 G T, (AGC) 5 G G, UBC 810, (GA) 9C, UBC 807, UBC 811, (GT) 7CA and (GA) 9A commercialized by UBC (the University of British Columbia) were used (Sheidai et al., 2012). Using a 25 μL volume containing 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany); 50 mM KCl; 10 mM Tris-HCl buffer at pH;8 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of each primer, polymerase chain reaction (PCR) was implemented. The reactions were amplified in Techne thermocycler (Germany)
2.3.2. Cp-DNA sequences analyses To investigate the species relationship like Networking and Bayesian approach, several phylogenetic methods were applied. MUSCLE program was applied to align the intron in the gene for ribosomal protein L16 (rpL16) first; then, it was used to examine the
Table 1 Dracocephalum, Lallemantia and Ziziphora species studied their localities and voucher numbers. No.
Province
Locality
Voucher number
Number of samples for ISSR study
1 2 3 4 5 6 7 8 9 10 11 12 13 14
D. kotschyi D. thymiflorum D. multicaule D. aucheri D. moldavica D. subcapitatum D. lindbergii L. peltata L. canescens L. baldschuanica L. iberica L. royleana Z. tenuior Z. persica
Mazandaran Mazandaran Qazvin Tehran Orumiyeh Khorasan Khorasan Alborz Qazvin Khorasan Zanjan Khorasan Qazvin Qazvin
HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU HSBU
7 6 5 9 5 3 5 9 5 6 5 8 – –
201213 201214 201215 201216 201217 201218 201219 201209 201200 201208 201210 201212 201220 201221
2
MH568733 MH453498 MH453499 MH453500 MH453497
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appropriate nucleotide substitution model as carried out in molecular evolutionary genetics analysis (MEGA) 7. Program (Tamura et al., 2012). Splits Tree4 program and BEAST software v1.6.1 (Drummond et al., 2010a, b) were used for networking and Bayesian analysis, respectively. 2.3.3. Time of species divergence To estimate divergence time in the studied species, Chloroplast DNA sequences were applied and for performing analyses, BEAST v1.6.1 was used (Drummond et al., 2010a, b). To have the initial xml files for BEAST, Bayesian evolutionary analysis utility version (BEAUti) v1.6.1 was used (Drummond et al., 2010a, b). The Yule tree prior is broadly known for providing the best-fit model for trees defining the relationships between various species that is capably of describing the net speciation rate (Drummond et al., 2010a, 2010b; Nee, 2006). For the MCMC analyses, the chain length was 10000000. 10000 trees were put for the analyses following the removal of 100 trees representing the burn-in. The BEAUti xml file was run in BEAST v1.6.1 (Drummond et al., 2010a, 2010b). Since there was were not any fossils for the target species, a rate of evolution of the plastid sequence (u = 1.0 × 10 −9 s s-1 year-1; Zurawski et al., 1984; Minaeifar et al., 2016) was considered that was entered in the option of molecular clock model in BEAUti v1.6.1. The normal distribution (Mean = 0, St. dev = 1) was used for priors. To test the sampling and convergence, Tracer v.1.5 was applied (Drummond and Rambaut, 2007). To explain the phylogenetic results generated by BEAST to form a single ‘target’ tree (maximum clade credibility tree (MCC)) including summary statistics, Tree Annotator v1.6.1 was applied. To generate the annotated BEAST (MCC) tree, FigTree v1.3.1 (Rambaut, 2009) was used. To analyze the biogeography, Cp-DNA data of the present study was used.
Fig. 1. ISSR profiles of 13 accessions of Dracocephalum using the primer UBC811. L: represent molecular weight size marker (100 kb ladder). N: negative control. The numbers represent different accessions according to Table 1.
polymorphism (44.23%) among the studied Dracocephalum species (Table 3). It has a wide geographical distribution in the country and occurs in Northern Provinces of Gorgan, Mazandaran, and Gilan, as well as in the Northeast Province of Azarbayejan and central Iran (Semnan and Tehran Provinces). Two species of D. thymiflorum and D. aucheri had almost similar level of genetic polymorphism. D. thymiflorum occurs in Mazandaran, Gilan, and Azarbayejan Provinces, and D. aucheri grows in Mazandaran, Tehran, Semnan, and Ghazvin. Dracocephalum multicuale had the lowest degree of genetic variability. It has also a confined geographical distribution and occurs only in two provinces of Ghazvin and Azarbayejan.
2.3.4. Biogeography The Bio- and phylogeographic markers are specially DNA sequences, yet, recently fingerprinting techniques, like simple sequence repeats (SSRs), amplified fragment length polymorphism (AFLPs), randomly amplified polymorphic DNA (RAPDs), and ISSRs, are also often used for phylogeographic investigations, because of the generally high levels of variability retrieved (Clausing et al., 2000; Hess et al., 2000; Pleines et al., 2009). To analyze biogeography, Reconstruction Ancestral State in Phylogenies program (RASP) programs was used (Yu et al., 2015). To infer ancestral state using statistical dispersal-vicariance analysis (SDIVA), RASP is usually applied. In the present study, both binary markov chain monte carlo (MCMC) and S- DIVA methods of RASP were applied for analyses. And based on ancestral of species distribution, the route of these species in the country was reconstructed.
3.2. Monophyly versus paraphyly in Dracocephalum WARD diagram presented previously based on ISSR data revealed that out-group species of Lallemantia are scattered among Dracocephalum species (Fig. 2). This indicated close affinity between these two genera and that they are not monophyletic. Close relationship between the species in both genera was supported by pair-wise AMOVA (Table 4) and the results did not show significant molecular difference between these species (FST = 0.17, P = 0.01). Therefore, although we obtained well delimited species based on K-Means clustering, they do not show significant molecular difference. However, when we compared the pooled data in each genus, AMOVA produced significant molecular difference; this indicates some degree of genetic differentiation between Dracocephalum and Lallemantia. This is further supported by K-Means clustering of both genera as the test produced optimal k = 2. However, in a phylogenetic context, these two genera are paraphyletic as evidenced by WARD analysis (Fig. 2). The representative species of Lallemantia were scattered among Dracocephalum species. Phylogenetic relationships of the studied species are provided in Bayesian tree of BEAST and TCS network (Figs. 3 and 4). The out-group species of Ziziphora formed a separate clade while the other species in Dracocephalum and Lallemantia were placed intermixed to some extent. Dracocephalum subcapitatum (Kuntze) Lipsky, D. kotschyi and D.
3. Results 3.1. Species delimitation and genetic diversity analyses by ISSR markers Out of a total of 8 different primers screened, only 6 primers ((CA) 7 G T, UBC 810, (GA) 9C, UBC 811, (GT) 7CA and (GA) 9A) produced reproducible and polymorphic bands for all species representing the taxa studied (Fig. 1). Clustering and network analyses of the studied species from Dracocephalum and Lallemantia (out-group taxa) produced similar groupings; therefore, only WARD is presented here (Fig. 2). The specimens of Dracocephalum species were almost delimited in a single cluster or group. Moreover, K-Means (Table 2) clustering recognized all 9 species (D. kotschyi, D. multicaule, D. thymiflorum, D. aucheri, and 5 species of Lallemantia) based on ISSR data, therefore the species rank is confirmed and they are delimited based on ISSR molecular markers. Dracocephalum kotschyi showed the highest degree of genetic 3
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Fig. 2. WARD diagram in Dracocephalum species based on cp-DNA. Table 2 K-Means clustering of the studied species.
Table 4 Paired-sample AMOVA between Dracocephalum and Lallemantia species.
k
SSD(T)
SSD(AC)
SSD(WC)
r-squared
pseudo-F
AIC
1 2 3 4 5 6 7 8 9&*
274.423 274.423 274.423 274.423 274.423 274.423 274.423 274.423 274.423
0.000 37.525 68.123 96.68 119.663 139.368 157.034 174.590 189.923
0.000 237.371 206.300 178.356 154.760 135.056 117.389 99.833 84.500
0.000 0.135 0.248 0.350 0.436 0.508 0.572 0.636 0.692
0.000 3.746 3.797 3.950 4.059 4.128 4.263 4.497 4.776
63.438 62.022 60.943 59.973 59.378 59.258 59.419 59.451 59.895
Pop1 Pop2 Pop3 Pop4 Pop5 Pop6 Pop7 Pop8 Pop9 a
* Best clustering according to Calinski & Harabasz' pseudo-F: k = 9. & Best clustering according to Bayesian Information Criterion: k = 9.
N
Na
Ne
I
He
uHe
%P
D. D. D. D.
10 10 10 7
0.885 0.683 0.467 0.673
1.160 1.175 1.098 1.192
00.174 0.164 0.090 0.168
0.107 0.107 0.058 0.111
0.113 0.113 0.061 0.120
44.23 33.65 22.12 32.69
kotschy thymiflorum multicuale aucheri
Pop2
Pop3
Pop4
Pop5
Pop5
Pop7
Pop8
Pop9
– 0.096 0.121 0.085 0.082 0.117 0.109 0.094 0.096
0.306 – 0.114 0.091 0.092 0.092 0.107 0.096 0.103
0.416 0.420 – 0.095 0.106 0.100 0.094 0.102 0.094
0.320 0.537 0.554 – 0.113 0.101 0.108 0.092 0.097
0.494 0.631 0.633 0.781 – 0.110 0.091 0.110 0.102
0.222 0.456 0.440 0.393 0.511 – 0.110 0.105 0.123
0.405 0.546 0.548 0.650 0.656 0.420 – 0.095 0.120
0.522 0.582 0.655 0.752 0.882 0.497 0.671 – 0.091
0.531 0.722 0.753 0.804 0941 0.643 0.652 0.936 –
Above diagonal : FST value, bellow diagonal : P value.
aucheri were placed close to each other. These species along with Lallemantia baldschuanica Gontsch., L. iberica (M.Bieb.) Fisch. & C.A.Mey., L. canescens (L.) Fisch. & C.A.Mey., and D. moldavica formed the second major clade. Dracocephalum multicuale and D. thymiflorum showed close affinity and comprised a single clade. They joined the afore-mentioned clade with some distance. Lallemantia peltata (L.) Fisch. & C.A.Mey., L. royleana (Benth.) Benth., and D. lindbergii Rech.f., showed affinity and formed the forth clade, separated from the other studied species. The method of templeton (TCS) haplotype network supported BEAST phylogenetic relationship, and revealed the presence of three main groups (Figs. 3 and 4). The first group was formed by D. subcapitatum, D. kotschyi, D. multicuale and D. thymiflorum. The second haplotype group was composed of L. peltata, L. royleana, and D.
Table 3 Genetic diversity parameters in the studied Dracocephalum species. Species
Pop1
a
N: No of plants studied. Na: No. of alleles. c Ne : Effective No. of alleles. d He: Gene diversity. e UHe : Unbiased gene diversity. f %P: Polymorphism percentage. b
4
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Fig. 3. BEAST chronogram of the studied species based on cp-DNA.
Dracocephalum occurred between 2–10 MYA. RASP analysis revealed that the most probable ancestral area of Dracocephalum species distribution in Iran is Mazandaran province located in the northern Iran. Two species of D. kotschyi and D. thymiflorum occurred in this province (No figure). Ghazvin province is the second probable ancestral area of Dracocephalum and it is occupied by D. multicuale. The more recent areas of the country occupied by Dracocephalum species are Tehran and Khorasan provinces. 3.4. HGT analysis The reticulogram produced by T-REX program showed horizontal gene transfer (HGT) between D. moldavica and D. subcapitalum, and also between D. moldavica and both L. iberica and L. canescens (Fig. 5). The HGT obtained is in agreement with inter-mixed positions obtained for the species in these two genera in both ISSR and Cp-DNA trees. HGT is the probable reason for the occurrence of common shared alleles in
Fig. 4. TCS haplotype network of cp-DNA.
lindbergii; while the other studied species formed the third major haplotype group. Further evidence in support of close genetic relationship between species in Dracocephalum and Lallemantia was obtained by AMOVA of Cp-DNA sequences. When AMOVA was performed among members of three genera viz. Dracocephalum, with those of out-groups Lallemantia and Ziziphora, it produced no significant molecular difference (Phi pt = 0.01, P .0.05). Nevertheless, when it was performed between combined Dracocephalum + Lallemantia with species in Ziziphora, a significant molecular difference was obtained (Phi pt = 0.17, **P < 0.01). 3.3. Time of divergence and ancestral area The chronogram of BEAST (Fig. 3) suggests that Ziziphora, Lallemantia and Dracocephalum studied species become diverged 30–80, while the more recent species divergence within Lallemantia and
Fig. 5. HGT tree showing gene flow among the studied Dracocephalum. 5
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Declaration of Competing Interest
these two genera, which in turn brings about their genetic similarity.
The authors declare no conflicts of interest.
4. Discussion
Acknowledgment
The sixth largest family of flowering plants with economic value is Lamiaceae family. Seven subfamilies were determined by Harley et al. (2004) within Lamiaceae. This classification influenced largely by morphological features and recent molecular reports (Drew and Sytsma, 2012). Apparently, Lamiaceae has numerous paraphyletic and genera that have not been investigated; for instance, Bendiksby et al. (2011) suggested the transfer of species to put the monophyly in genus Lamium L. and Otostegia Benth.. They reported that numerous genera are nonmonophyletic including Ballota s.str., Lagopsis (Bunge ex Benth.) Bunge, Leonotis (Pers.) R.Br., Leonurus L., Leucas R.Br., Microtoena Prain., Phlomoides Moench., Sideritis L., Stachys L., and Thuspeinanta T.Durand. Based on this study, Dracocephalum is a paraphyletic genus and this is consistent with the previous results (Drew and Sytsma, 2012). Lamiaceae genera developed along with extensive inter-specific hybridization and polyploidy events; thus understanding the species relationship will not be easy, as there is incongruence between phylogenetic trees from diverse sets of genes (Trusty et al., 2004; Salmaki et al., 2013). The present study highlighted the horizontal gene transfer (HGT) between D. moldavica and D. subcapitalum, and also between D. moldavica and both L. iberica and L. canescens. In our study we found that ISSRs can delimit Dracocephalum species. These dominant molecular markers are known to delimit different plant taxonomic groups like Salvia L., Cirsium Mill., Olive L., and Tamarix L., etc. (see for example, Sheidai et al., 2012, 2013; Sheidai et al., 2014; Ijbari et al., 2014; Safaei et al., 2016). Sonboli et al. (2011) studied Dracocephalum species and reported that RAPD markers can be used in species delimitation. The split between Phrymaceae + Linderniaceae and Lamiaceae, dates from 60 to 107 MYA (Drew and Sytsma, 2012). Lamiaceae are not well characterized in the fossil record (Harley et al., 2004), however Kar (1996) recognized the fossil Ocimum L., which is within Nepetoideae, and dates back to 49-MYA and the Early Eocene. In the EarlyMiddle Oligocene, A fossil fruit of Melissa was described that dates back to 28.4 MYA (Reid and Chandler, 1926; Martínez-Millán, 2010). Until now, Drew and Sytsma (2012) have used Cp-DNA to specify divergence time in Nepetoideaebased on fossil calibration. They believed that subfamily Nepetoideae extended around 57 Ma at the Paleocene-Eocene border. The development of the extant members of tribe Mentheae took place 46 MYA in the mid-Eocene; yet, all the evolution of the sub-tribes was by the Eocene-Oligocene border (ca. 34 MYA). All sub-tribes underwent generic differentiation in late Miocene or early Pliocene except Menthinae. Similarly, generic differentiation of Old World (primarily Mediterranean) Menthinae also happened during the late Miocene; yet, the North and South American sub-clade of Menthinae underwent differentiation far more recent in the Pliocene (Drew and Sytsma, 2012). The present study is in agreement with the findings of Drew and Sytsma (2012) and suggests that Ziziphora, Lallemantia and Dracocephalum studied species become diverged 30–80 mya, while the more recent species divergence within Lallemantia and Dracocephalum occurred between 2–10 MYA.
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