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PHYLOGENETIC RELATIONSHIP AMONG SELECTED SPECIES OF LAMIACEAE INFERRED FROM CHLOROPLAST RPS14 GENE.

Byline: A. Malik, T. Shahzad, S. Arif, W. Akhtar and T. Mahmood

Key words: Lamiaceae, Iterative Threading Assembly Refinement, rps14 gene, Pairwise distance, Genetic diversity.

INTRODUCTION

The Lamiaceae is family of flowering plant in the order Lamiales that comprised of annual or perennial herbs and shrubs with opposite leaves. The stem is mostly square and leaves when grinded releases pleasant odors (Metcalfe and Chalk, 1950). The Lamiaceae included 240 genera, more than 7,000 taxa in the World and seven subfamilies namely Viticoideae, Ajugoideae, Nepetoideae, Scutellarioideae, Prostantheroideae, Symphorematoideae and Lamioideae (Harley et al., 2004), now new subfamilies are comprised of Peronematoideae, Tectonoideae, Cymarioideae, Callicarpoideae and Premnoideae (Li et al., 2016; Li and Olmstead, 2017). The Lamiaceae is found in various altitudes and habitats varying from North Eastern Asia to Hawaii, North Pole areas to the Himalayas, America, Africa and Australia (Erdem et al., 2017). The Lamiaceae is economically important for various timber trees like Tectona, many species are medicinal and utilized as culinary herbs.

The Lamiaceae species are also used for horticultural purposes, in perfumery and nectar producing species that produce high-quality honey (Harley, 2012). The molecular phylogenetic studies are conducted to determine the relationship among organisms or genes by comparing the DNA or protein sequences (Patwardhan et al., 2014). The chloroplast genome (cp) comprised of 120-130 genes with conserved gene content and gene order (Lei et al., 2016). The cp genome experienced selection pressure during course of evolution and recent phylogenetic study indicate numerous positive selection genes such as psaA, atpA, atpB, rbcL, rpI20 and ndhI genes in Urophysa (Xie et al., 2018).The cp genome has uniparental inheritance, moderate evolutionary rate and conserved structure that makes cp genome applicable for DNA barcoding and phylogenetic studies (Luo et al., 2016: Li et al., 2015: Dong et al., 2018; Yang et al., 2018).

A wide range of molecular markers have been utilized to study phylogeny of Lamiaceae such as matK, ndhF, rbcL, rps16 and trnL-F (Li et al., 2016) ITS and ndhF (Steane et al., 2004), trnL intron, trnL-trnF and rps16 (Paton et al., 2004), on the basis of four cpDNA regions ycf1, ycf1-rps15 spacer, trnL-F and rpI32-trnL (Drew and Sytsma, 2012). The plastid genes psaA and psaB in rice encoded the two apoproteins of P700 chlorophyll a protein complex of photosystem I reaction center and rps14 gene that encodes the ribosomal protein S14 are organized into a transcription unit (Chen et al., 1992). The rps14 gene has been reported from the cp genome of N. tabacum (Wakasugi et al., 1998). In the past, rps14 gene has been used for the assessment of phylogenetic relationship among Plantago species, Mentha, Citrus species and date palm varieties (Saeed et al., 2011; Jabeen et al., 2012; Wali et al., 2013; Akhtar et al., 2014).

In the past, phylogenetic relationship among various genera of Lamiaceae such as Clerodendrum, Vitex, Tectona, Gmelina, Callicarpa and Caryopteris had not been focused based on rps14 gene. Therefore, the hypothesis of present study is an assessment of genetic diversity and phylogenetic relationship among selected Lamiaceae species based on rps14 gene and structural validation of rps14 protein via Ramachandran plots.

MATERIALS AND METHODS

Plant Collection: The leaves of plant species were collected from Islamabad, Rawalpindi, Lahore and Faisalabad regions of Pakistan and stored at 4 AdegC for future purpose (Table 1). The correct taxonomic placement of plant species was done by following the International Plant name Index the Royal Botanic Gardens Kew, UK (http://plantsoftheworldonline.org/).

Table 1: List of selected species of family Lamiaceae.

S/N###Plant Species###Cities###Nature of Plant Species

###1###Callicarpa macrophylla###Faisalabad###Shrub

###2###Tectona grandis###Lahore###Tree

###3###Caryopteris odorata###Islamabad###Shrub erect or suberect

###4###Gmelina philippensis###Islamabad###Straggling or scandent spinose shrub

###5###Clerodendrum umbellatum###Islamabad###Shrub, climbing to suberect

###6###Vitex trifolia###Islamabad###Shrub or small tree

###7###Clerodendrum calamitosum###Islamabad###Small to medium size woody shrub

###8###Clerodendrum indicum###Islamabad###Shrub

###9###Volkameria inermis###Islamabad###Erect to scandent or trailing ever green shrub

10###Vitex agnus castus###Islamabad###Erect shrub

DNA isolation and Primer designing: The genomic DNA was isolated from stored leaves of Lamiaceae by CTAB method (Richards et al,. 1997) with little modifications. The extraction buffer 2% CTAB (2.4 g of Tris HCl, 1.16 g of EDTA, 16.3 g of Nacl and 4 g of CTAB) was prepared in 200 ml distilled water. The plant material (0.4 g) was crushed in 1500 ul pre-heated CTAB at 65 AdegC along with 15 ul of mercaptoethanol. The homogenized mixture was transferred to eppendorf tubes, incubated at 65 AdegC for 1 hour and centrifuged at 12,000 rpm for 15 minutes. The supernatant was transferred to a new eppendorf and washed 3-4 times with equal volume of Chloroform isoamyl alcohol (24:1) and centrifuged at 12,000 rpm for 15 minutes. The pre-chilled isopropanol was added to clear supernatant and tubes were left overnight at-20 AdegC, then sample was centrifuged and supernatant was discarded. The pellet was washed with cold 70% ethanol and air dried at room temperature for 1 hour.

The air-dried pellet was dissolved in TE (Tris EDTA) buffer containing RNase (7 ul of RNase added in 1mL buffer) and incubated at 37 AdegC for 10 minutes. The obtained DNA was stored at-20 AdegC. The DNA isolation and PCR amplification were performed in the Plant Molecular biology and Biochemistry Lab situated in Quaid-i-Azam University Islamabad. The primers were designed for rps14 gene through online primer 3 (version 4.10) (http://bioinfo.ut.ee/primer3/) based on cp genome of N. tabacum available in NCBI Genbank (https://www.ncbi.nlm.nih.gov). The primer sequence is mentioned below;

rps14 F: 5'-ATGGCAAGGAAAAGTTTGATTC-3'

rps14 R: 5'-TTACCAACTTGATCTTGTTGCTCCT-3'

PCR amplification conditions and sequencing: The following conditions were used for the amplification of rps14 gene in PCR Multi Gene thermal cycler (Labnet). The initial denaturation at 94 AdegC for 5 minutes was followed by 35 cycles of denaturation at 94 AdegC for 30 seconds, annealing in the range of 45-51 AdegC for 1 minute, extension at 72 AdegC for 1 minute and final extension at 72AdegC for 20 minutes. The amplified PCR product was purified by Gene JET PCR Purification Kit and sequenced from the Beijing Genomic Institute, Shenzhen, China.

Sequence analysis: The rps14 gene sequences of Lamiaceae species were uploaded and BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed one by one in query form for comparison with already reported sequences in Genbank. The nucleotide sequences were cleaned and aligned with the help of online server JustBio (https://www.justbio.com/hosted-tools.html). The nucleotide sequences were converted into amino acid sequences through EXPASY-Translate tool (https://Web.expasy.org/ translate/). The sequences data of rps14 gene of all ten Lamiaceae species were submitted to the Genbank for accession numbers (https://www.ncbi.nlm.nih.gov/home/submit/) (Table 2).

Table 2: Accession numbers and Nucleotide composition of rps14 gene from selected species of family Lamiaceae.

S/N###Plant species###Accession Number###T(U)###C###A###G###Total

###1###Vitex agnus castus###MH382179###23.8###18.2###34.0###24.1###303

###2###Callicarpa macrophylla###MH382180###24.8###18.8###34.3###22.1###303

###3###Tectona grandis###MH382183###24.4###18.5###34.7###22.4###303

###4###Caryopteris odorata###MH382184###24.1###18.2###34.0###23.8###303

###5###Gmelina philippensis###MH382186###24.4###18.2###36.0###21.5###303

###6###Clerodendrum umbellatum###MH382188###24.1###19.5###34.0###22.4###303

###7###Vitex trifolia###MH382190###23.8###18.2###34.0###24.1###303

###8###Clerodendrum calamitosum###MH382191###24.4###19.1###34.3###22.1###303

###9###Clerodendrum indicum###MH382192###24.4###19.1###34.7###21.8###303

10###Volkameria inermis###MH382194###24.4###19.1###34.0###22.4###303

###Average###24.3###18.7###34.4###22.7###303

Phylogenetic analysis: The aligned sequences were subjected to MEGA7 with Maximum Likelihood (ML) method and 1000 BS value was used for construction of phylogenetic tree (Kumar et al., 2016). The DNA sequences were aligned into FASTA format and uploaded in MEGA7 with nucleotide sequences, protein coding nucleotide sequence data and standard genetic code. The gaps/missing data treatment was completely deleted for calculation of Pairwise distance, Tajima's Test of Neutrality and Substitution patterns. In MEGA7 the selection tool was used for calculation of Tajima's Test of Neutrality. The pairwise distance was calculated by Tamura-Nei model with substitution of transitions and transversions. The substitution Matrix (ML) values were determined by Tamura-Nei model and Maximum Likelihood method.

Statistics: The Tajima's D statistics was used for detection of selection using rps14 gene sequences in MEGA7 (Tajima, 1989). The Tajima's D can be described as if D =0 observed data =expected = no selection, D = negative = D < 0 observed 0 observed > expected= More average heterozygosity as compared to segregating sites showed balancing selection. The best DNA model (ML) was determined by MEGA7 (Kumar et al., 2016) in which the lowest BIC (Bayesian information criterion) scores indicated the substitution pattern best. The model with best substitution pattern was then used to build the substitution pattern estimation (ML), Pairwise distance and phylogenetic tree.

The statistical support of phylogenetic tree was determined by Bootstrapping method performed with 1000 bootstrap replications in MEGA7 (Kumar et al., 2016).

The statistics used in the I TASSER was C-score, Z-score and TM-scores for 3D protein models. The threading alignment of 3D protein models was denoted by Z. The Z is measured as the difference between the raw and average scores in the unit of standard deviation. The Normalized Z-score > 1 indicates good alignment. The C-score (Confidence score) is measured as significance of threading template alignments and the convergence parameters of the structure assembly simulation. The value of C-score was in the range of-5 to 2 that indicates higher confidence and more reliable prediction. The TM-score (Template based score) measuring the structural similarity between two structures. A TM-score has value ranges from (0-1) and TM-score > 0.5 indicates a model of similarity of protein to the template (https:// Zhanglab.ccmb.med. umich.edu/I-TASSER).

The 3D models was validated by RAMPAGE that shows the scores of allowed region, favored region and outlier region in which the < 2% amino acid in the outlier region of different Lamiaceae species showed good protein models (http://mordred.bioc.cam.ac.uk/ ~rapper/rampage.php).

Protein structure prediction and validation: I-TASSER is online software used for prediction of 3D protein structures (https:// Zhanglab.ccmb.med.umich. edu/I-TASSER). The validation of 3D Protein models was done by RAMPAGE (http://mordred.bioc.cam.ac.uk /~rapper/ rampage.php). The Ramachandran plots formed by uploading Pdb files of 3D protein models on RAMPAGE. The Ramachandran plots were used for structural validation of 3D protein structures and to visualize the conformation of dihedral angle I and I" of amino acid residues present in proteins.

RESULTS

Amplification of rps14 gene: The target gene rps14 was amplified by specific forward and reverse primers. The band was observed under UV light in Dolphin-Doc Plus gel documentation system. The amplification process gave clear bands of 303 bp for Lamiaceae species (Figure 1).

Sequence analysis: The BLASTn comparisons of chloroplast rps14 gene sequence with already reported sequence revealed 99% sequence identity with chloroplast genome of Vitex negundo (Accession number # MF678773).

Estimation of nucleotide composition: The average of nucleotide number was T (U) = 24.3, C= 18.7, A = 34.4 and G = 22.7. The A has highest average nucleotide number of 34.4 and C has lowest average nucleotide number of 18.7. The total average number of all nucleotides was 303 (Table 2).

Tajima's neutrality test: In Lamiaceae ten numbers of sequences gave 41 segregation sites (S) and nucleotide diversity (I) of 0.056912. The low nucleotide diversity of 0.056912 revealed close genetic relationship among studied Lamiaceae species. The positive value of Tajima's D statistics was 0.922432 which showed more haplotypes that increase the chance of average heterozygosity than segregating sites and balancing selection would operate (Tajima, 1989) (Table 3).

Estimation of substitution matrix (ML): The lowest BIC score of 1790.792 indicated TN93 model that was Tamura-Nei (1993) model so the substitutions pattern and rates were determined through Tamura-Nei (1993) model. The relative values of instantaneous (r) should be included when calculating them, for easiness the sum of (r) values is made equal to 100. The values of transitional substitution were higher than transversionsal substitution. The nucleotide frequencies for Lamiaceae were A = 34.39%, T/U = 24.26%, C = 18.68% and G = 22.67%. The calculation of ML values for Lamiaceae were done by computing the tree topology. The ML Log for Lamiaceae was-863.363 (Table 4). The statistical result of lowest BIC score showed that in Tamura-Nei model there were different nucleotide frequencies. So, substitution matrix (ML) values estimated by Tamura-Nei model also have different nucleotide frequencies (Table 5).

Table 3: Tajima's neutrality test values based on rps14 gene of Lamiaceae.

No. of sequences###Number of segregating###Ps###T###Nucleotide diversity###Tajima's test

###"m"###sites "S"###"I"###Statistics "D"

###10###41###0.135314###0.047831###0.056912###0.922432

Table 4: Maximum likelihood values of transitional (bold) and transversionsal substitutions (italics) of nucleotides of rps14 gene for Lamiaceae.

###A###T/U###C###G

###A###-###4.02###3.09###18.28

###T/U###5.69###-###9.09###3.75

###C###5.69###11.80###-###3.75

###G###27.72###4.02###3.09###-

The nucleotide frequencies for Lamiaceae were A = 34.39%, T/U = 24.26%, C = 18.68% and G = 22.67%. The calculation of ML values for Lamiaceae were done by computing the tree topology. The ML Log for Lamiaceae was-863.363.

Calculation of Pairwise distance: In Lamiaceae the value of genetic diversity lies in the range of 0.013 to 0.089 with overall mean distance of 0.060 for rps14 gene. The low mean distance value of 0.060 show close genetic relationship among Lamiaceae species and low genetic diversity among them on the basis of rps14 gene (Table 6).

Phylogenetic tree: The phylogenetic tree was built through MEGA7 based on rps14 gene sequences (Kumar et al., 2016). The dendrogram revealed little genetic distance of 0.0100 that showed close genetic relationship among Lamiaceae species. The phylogram was divided in two clusters and three groups denoted by cluster I, cluster II, group I, group II and group III. The cluster I consisted of C. umbellatum, C. odorata, C. indicum, C. calamitosum and Volkameria inermis. In cluster I, C. odorata showed close phylogenetic relationship with C. indicum, C. calamitosum and Volkameria inermis with BS value of 58%. The C. indicum, C. calamitosum and Volkameria inermis also showed close phylogenetic association among them with BS value of 51%. The C. umbellatum showed recent evolution with small branch length of 0.021. While C. odorata were earliest evolved member with more branch length of 0.039. The C. odorata also depicted close relationship with C. umbellatum in the same cluster.

In group I, C. calamitosum and Volkameria inermis also revealed close phylogenetic relationship between them. The Cluster II included V. agnus castus. V. trifolia, C. macrophylla, T. grandis and G. philippensis and divided into group II and group III. In group II, C. macrophylla and G.philippensis revealed close relationship and grouped together in the cluster II. The G. philippensis was recently evolved member with branch length of 0.017. While C. macrophylla were earliest evolved with branch length of 0.021. In group III, V. agnus castus and V. trifolia depicted close phylogenetic relationship with BS value of 99%. The C. macrophylla and T. grandis also showed close relationship among them in the cluster II. The C. macrophylla showed earliest evolution with branch length of 0.021.While T. grandis was recently evolved member with branch length of 0.010.

The G. philippensis and T. grandis revealed close relationship and were grouped together in the cluster II. The V. trifolia and C.macrophylla also showed close phylogenetic association among them. The V. agnus castus and T. grandis showed close relationship and were grouped together in the cluster II. The V. trifolia showed phylogenetic association with G. philippensis in the same cluster based on rps14 gene. The V. agnus castus and G. philippensis revealed close relationship among them. The V. agnus castus showed recent evolution with branch length of 0.003 while G. philippensis showed the earliest evolution with branch length of 0.017. In Cluster II, V. trifolia and T. grandis revealed close relationship among them (Figure 2).

Analysis of 3D proteins structures: The 3D protein models were constructed through I-TASSER and top five models of 3D protein structures for each species were predicted. The confidence of each model was calculated on the basis of confidence score (C-score). The value of C-score was in the range of-5 to 2. The 3D protein model having higher C-score was indication of higher confidence and more reliable prediction. The rps14 protein contains 100 amino acid residues and 3D protein models consists of the alpha helices, beta strands and coils the number of alpha helices are more than beta strands and coils. The predicted solvent accessibility of 3D protein model is from hydrophilic to hydrophobic. The C-score, Z-score and TM-score is good in all 3D protein model of Lamiaceae(Table 7).

Protein structure validation: The Ramachandran plots consisted of quadrant of different colors. The dark blue and dark orange areas indicated the favored region. Whereas the light blue and light orange were allowed region. The unshaded areas showed outlier region (Figure 3). In Lamiaceae [greater than or equal to] 81 to a$? 88 residues in favored region, [greater than or equal to] 8 to a$? 13 in allowed region and a$? 2 to [greater than or equal to] 3 in outlier region. The protein models of Gmelina philippensis, Clerodendrum umbellatum and Clerodendrum calamitosum were considered to be good protein models as they had a$? 2% amino acid residues in the outlier region (Table 8).

Table 5: Maximum Likelihood fits of 24 different nucleotide substitution models.

###TN93###22###1790.792###1664.75###-863.363###n/a###n/a###2.09###0.34###0.24###0.187###0.277###0.040###0.031###0.183###0.057###0.091###0.038###0.057###0.118###0.038###0.277###0.040###0.031

###6###4###3

K2+G+I###20###1791.518###1671.47###-815.596###0.06###0.29###2.92###0.25###0.25###0.250###0.250###0.032###0.032###0.186###0.032###0.186###0.032###0.032###0.186###0.032###0.186###0.032###0.032

###1###3###0###0

###T92+G###20###1792.723###1672.67###-816.198###n/a###0.05###2.47###0.29###0.29###0.207###0.207###0.041###0.029###0.148###0.041###0.148###0.029###0.041###0.211###0.029###0.211###0.041###0.029

###6###3###3

###K2+G###19###1792.750###1678.62###-820.220###n/a###0.05###2.51###0.25###0.25###0.250###0.250###0.036###0.036###0.179###0.036###0.179###0.036###0.036###0.179###0.036###0.179###0.036###0.036

###0###0

HKY+G+I###23###1797.672###1658.66###-806.648###0.61###0.27###2.73###0.34###0.24###0.187###0.227###0.033###0.025###0.166###0.046###0.136###0.046###0.031###0.177###0.031###0.251###0.033###0.025

###4###4###3

HKY+G###22###1799.067###1667.04###-811.354###n/a###0.05###2.40###0.34###0.24###0.187###0.227###0.036###0.028###0.160###0.051###0.132###0.033###0.051###0.171###0.033###0.242###0.036###0.028

###5###4###3

TN93+G+I###24###1804.138###1660.14###-805.873###0.05###0.25###2.83###0.34###0.24###0.187###0.227###0.033###0.025###0.205###0.046###0.094###0.030###0.046###0.122###0.030###0.311###0.033###0.025

###5###8###4###3

TN93+G###23###1804.236###1666.22###-809.931###n/a###0.05###2.53###0.34###0.24###0.187###0.227###0.035###0.027###0.202###0.050###0.087###0.050###0.033###0.112###0.033###0.307###0.305###0.027

###8###4###3

###JC+G###18###1826.785###1718.71###-841.246###n/a###0.05###0.50###0.25###0.25###0.250###0.250###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083

###9###0###0

GTR+G+I###27###1828.103###1666.16###-805.831###0.58###0.25###2.82###0.34###0.24###0.187###0.227###0.033###0.028###0.204###0.047###0.095###0.027###0.051###0.123###0.029###0.310###0.029###0.024

###6###4###3

GTR+G###26###1828.151###1672.19###-809.863###n/a###0.05###2.53###0.34###0.24###0.187###0.277###0.037###0.025###0.202###0.052###0.087###0.029###0.047###0.113###0.039###0.306###0.031###0.032

###4###4###3

JC+G+I###19###1828.463###1714.40###-853.014###0.06###0.29###0.50###0.25###0.25###0.250###0.250###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083

###5###0###0###0

###T92+I###20###1855.601###1735.55###-847.637###0.43###n/a###2.15###0.29###0.29###0.227###0.227###0.046###0.032###0.143###0.046###0.143###0.032###0.046###0.202###0.032###0.202###0.046###0.032

###4###3###3

###K2+I###20###1858.367###1744.30###-853.028###0.43###n/a###2.15###0.25###0.25###0.250###0.250###0.040###0.040###0.171###0.040###0.171###0.040###0.040###0.171###0.040###0.171###0.040###0.040

###9###0###0

###HKY+I###19###1861.651###1729.62###-842.646###0.43###n/a###2.14###0.34###0.29###0.187###0.227###0.039###0.030###0.154###0.055###0.127###0.036###0.055###0.165###0.036###0.234###0.039###0.030

###8###4###3

###TN93+I###22###1865.632###1727.62###-839.625###0.43###n/a###2.14###0.34###0.24###0.187###0.227###0.039###0.041###0.170###0.059###0.105###0.041###0.059###0.137###0.041###0.244###0.041###0.032

###9###4###3

###JC+I###23###1866.390###1728.38###-841.007###0.43###n/a###2.21###0.25###0.25###0.250###0.250###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083

###2###0###0

###GTR+I###26###1890.274###1734.36###-840.925###0.43###n/a###2.21###0.34###0.24###0.187###0.227###0.035###0.030###0.190###0.050###0.086###0.055###0.037###0.112###0.042###0.289###0.040###0.035

###5###4###3

###T92###19###1894.234###1734.34###-849.732###0.43###n/a###2.21###0.29###0.29###0.210###0.210###0.048###0.035###0.140###0.048###0.140###0.035###0.048###0.193###0.035###0.193###0.048###0.035

###7###0###0

###K2###18###1891.812###1777.75###-869.751###n/a###n/a###2.09###0.25###0.25###0.250###0.250###0.040###0.040###0.169###0.040###0.169###0.040###0.040###0.169###0.040###0.169###0.040###0.040

###5###0###0

###HKY###21###1893.928###1785.86###-874.817###n/a###n/a###2.09###0.34###0.23###0.183###0.237###0.039###0.030###0.153###0.056###0.126###0.037###0.056###0.164###0.037###0.232###0.039###0.030

###2###1###9

T92+G+I###21###1897.839###1771.80###-864.748###n/a###n/a###2.08###0.34###0.34###0.210###0.210###0.037###0.026###0.155###0.037###0.155###0.026###0.037###0.220###0.026###0.220###0.037###0.026

###4###4###4

###JC###17###1903.084###1771.06###-811.225###0.61###0.27###2.89###0.25###0.25###0.250###0.250###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083###0.083

###2###0###0

###GTR###25###1923.993###1821.91###-893.858###n/a###n/a###0.50###0.34###0.23###0.183###0.237###0.036###0.031###0.182###0.051###0.091###0.039###0.058###0.118###0.042###0.276###0.041###0.035

###9###1###9

Models with lowest BIC scores (Bayesian information criterion) are considered to describe the substitution pattern best AICc (Akaike information criterion values, InL (Maximum Likelihood values) +G (discrete Gamma distribution) +I (invariant sites) f (nucleotide frequency) r (rate of base substitution) Assumed or estimated values of Transition/ Transversion bias (R) for each model. The sum of r values is equal to 1 for each model.

Abbreviation: GTR=General Time Reversible, HKY=Hasegawa-Kishino-Yano, TN93=Tamura-Nei Model, T92= Tamura 3-parameter, K2=Kimura 2-parameter, JC=Jukes-Cantor.

Table 6: Pairwise distance of rps14 gene of family Lamiaceae using MEGA7.

###Plant species###1###2###3###4###5###6###7###8###9###10

Callicarpa macrophylla###0.00

###Tectona grandis###0.037

###Caryopteris odorata###0.063###0.062

###Gmelina philippensis###0.038###0.034###0.077

Clerodendrum umbellatum###0.059###0.037###0.067###0.055

###Vitex trifolia###0.056###0.059###0.070###0.059###0.081

Clerodendrum calamitosum###0.041###0.055###0.056###0.070###0.056###0.081

###Clerodendrum indicum###0.059###0.059###0.067###0.066###0.052###0.081###0.037

###Volkameria inermis###0.052###0.059###0.056###0.073###0.066###0.081###0.038###0.048

###Vitex agnus castus###0.063###0.052###0.084###0.059###0.067###0.013###0.074###0.088###0.089

Table 7: Statistical analysis of rps14 protein models of Lamiaceae by I-TASSER.

S/N###Plant species###Z###C-###TM-###Description of Statistical values Good C score, Z

###Score###Score###Score###Score and TM Score for all species

1###Tectona grandis###3.53###0.91###0.892###Good alignment, higher confidence and best match with

###template

2###Vitex agnus castus###3.51###0.90###0.896###Good alignment, higher confidence and best match with

###template

3###Vitex trifolia###3.50###0.93###0.905###Good alignment, higher confidence and best match with

###template

4###Gmelina philippensis###2.79###0.96###0.900###Good alignment, higher confidence and best match with

###template

5###Callicarpa macrophylla###3.56###0.91###0.896###Good alignment, higher confidence and best match with

###template

6###Caryopteris odorata###3.16###0.93###0.888###Good alignment, higher confidence and best match with

###template

7###Volkameria inermis###3.54###0.94###0.899###Good alignment, higher confidence and best match with

###template

8###Clerodendrum indicum###3.06###0.89###0.882###Good alignment, higher confidence and best match with

###template

9###Clerodendrum###2.79###0.94###0.906###Good alignment, higher confidence and best match with

###umbellatum###template

10###Clerodendrum###3.25###0.92###0.872###Good alignment, higher confidence and best match with

###calamitosum###template

Table 8: Ramachandran scores of rps14 protein model of Lamiaceae by RAMPAGE.

###S/N###Plant species###Favored region###Allowed region###Outlier region

###1###Vitex agnus castus###88###8###2

###2###Callicarpa macrophylla###81###10###7

###3###Tectona grandis###85###9###4

###4###Caryopteris odorata###87###9###2

###5###Gmelina philippensis###85###13###0

###6###Clerodendrum umbellatum###85###13###0

###7###Viex trifolia###88###8###2

###8###Clerodendrum calamitosum###84###13###1

###9###Clerodendrum indicum###83###13###2

###10###Volkameria inermis###84###11###3

DISCUSSION

The present study reveals a close relationship of Caryopteris and Clerodendrum based on rps14 gene. This results are in congruent with the findings of Li et al. (2016) in which Caryopteris and Clerodendrum showed close relationship in the same clade of subfamily Ajugoideae based on matK, ndhF, rbcL,rps16 and trnL-F. The close relationship of Caryopteris with Clerodendrum was also studied on the basis of ndhF marker (Steane et al., 1997). The close relationship of C. indicum, C. calamitosum and V. inermis based on rps14 gene was also supported by (Steane et al., 1997, 1999 and 2004) based on cpDNA restriction site, ITS marker and cpDNA restriction site data, ITS and ndhF markers. Xiang et al. (2018) also reported close phylogenetic relationship of C. indicum, C. calamitosum and V. inermis based on matK, rbcL, trnL intron, trnL-F and rps16 markers.

The V. agnus castus and V. trifolia depicted close phylogenetic relationship based on rps14 gene which was in accordance with the findings of Li et al. (2016) in which Vitex agnus castus and Vitex trifolia showed close phylogenetic association based on matK, ndhF, rbcL, rps16 and trnL-F markers. Bramley et al. (2009) reported close relationship of V. agnus castus and V. trifolia based on combined analysis of ITS and ndhF sequences.

The present study indicated that C. macrophylla and T. grandis showed close relationship in the same cluster based on rps14 gene that was also studied by Paton et al. (2004) on the basis of trnL intron, trnL-F and rps16 markers. The close relationship was also seen by Steane et al. (2004) on the basis of ndhF sequences, Wagstaff et al. (1998) on the basis of combined analysis of rbcL and ndhF sequences. Scheen et al. (2010) studied the close relationship of C. macrophylla with T. grandis based on trnL-F regions and rps16 intron.

The close phylogenetic relationship of G. philippensis and T. grandis based on rps14 gene were also consistent with the findings of Paton et al. (2004) in which Gmelina showed close relationship with T. grandis on the basis of trnL intron, trnL-F and rps16, Xiang et al. (2018) based on matK, rbcL, trnL-F and rps16 molecular markers, Bendiksby et al. (2011) based on the cp genes matK, rps16, trnL intron and trnL-F spacer. Scheen et al. (2010) also studied the close relationship of Gmelina with T. grandis based on trnL-F regions and rps16 intron.

The T. grandis and V. agnus castus revealed close relationship based on rps14 gene was also supported by Li et al. (2016) based on matK, ndhF, rbcL, rps16 and trnL-F markers. Wagstaff et al. (1998) reported close phylogenetic relationship of T. grandis with V. agnus castus on the basis of rbcL and ndhF data sets. The V. trifolia showed phylogenetic association with G. philippensis in the same cluster in the present study that are similar to Bendiksby et al. (2011) on the basis of trnL-intron, trnL-F spacer, rps16 intron and matK, Paton et al. (2004) on the basis of trnL intron, trnL-trnF intergene spacer and rps16 intron. Scheen et al. (2010) also reported close phylogenetic association of V. trifolia and Gmelina based on the trnL-F regions and rps16 intron. The close association of V. trifolia and C. macrophylla on the basis of rps14 gene was supported by Paton et al. (2004) in which Vitex trifolia showed close relationship with Callicarpa on the basis of trnL intron, trnL-F and rps16.

The V. agnus castus and G. philippensis depicted close phylogenetic association and were grouped together in the same cluster on the basis of rps14 gene and comparable with the findings of Chen et al. (2014) in which V. agnus castus and G. philippensis showed close relationship in the same cluster based on ndhF and rbcL. The close association of Callicarpa and Gmelina in the same cluster based on rps14 gene was also reported by Scheen et al. (2010) on the basis of the trnL-F regions and rps16 intron. The V. trifolia revealed close relationship with T. grandis in the same cluster based on the rps14 gene was also studied by Scheen et al. (2010) based on the trnL-F regions and rps16 intron.

The present study showed lowest genetic diversity of (0.060) among Lamiaceae species based on rps14 gene. These results were comparable with diversity accessed by Talebi et al. (2019) in which low genetic diversity (0.063) was observed among Salvia nemorosa of family Lamiaceae collected from different parts of Iran and consists of several local populations based on ISSR marker. The method of conservation among Lamiaceae species are ex-situ conservation (Sun et al., 2019), as low genetic diversity in present study revealed ex-situ conservation that leads to diversification between Lamiaceae species. The result of the present study also supports the earlier work based on different DNA markers indicating that rps14 gene can be used for evaluation of the phylogenetic relationship and genetic diversity.

The present study shows that rps14 gene is a potential marker as it revealed the close relationship among Lamiaceae species and low genetic diversity which is indicative of the conservative trend among Lamiaceae species. The validation of rps14 protein by RAMPAGE also indicated good protein models in different Lamiaceae species.

Conclusion: The present study revealed the close genetic relationship and low genetic diversity among different Lamiaceae species based on rps14 gene showing their usefulness for the phylogenetic relationship and genetic diversity analysis among Lamiaceae species. The 3D protein structures formed by I-TASSER and its validation through RAMPAGE indicated good protein models.

Author's contribution: All authors have equal contribution data analysis, drafted and review the manuscript.

Acknowledgements: We would like to thank Dr. Amir Sultan, Dr. Muhammad Zafar and Dr. Mushtaq Ahmad for identification of plant species.

Conflict of interest: The authors declare that there is not any conflict of interest in the manuscript.

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Author:A. Malik, T. Shahzad, S. Arif, W. Akhtar and T. Mahmood
Publication:Journal of Animal and Plant Sciences
Geographic Code:9PAKI
Date:Jun 22, 2021
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