Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(4): 3-13, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1592 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Laleh Malekmohammadi, Masoud Sheidai, Farrokh Ghahrema- ninejad, Afshin Danehkar, Fahimeh Koohdar (2022). Avicennia genus molecu- lar phylogeny and barcoding: A mul- tiple approach. Caryologia 75(4): 3-13. doi: 10.36253/caryologia-1592 Received: March 02, 2022 Accepted: October 20, 2022 Published: April 28, 2023 Copyright: © 2022 Laleh Malekmo- hammadi, Masoud Sheidai, Farrokh Ghahremaninejad, Afshin Danehkar, Fahimeh Koohdar. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits 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. Avicennia genus molecular phylogeny and barcoding: A multiple approach Laleh Malekmohammadi1, Masoud Sheidai1,*, Farrokh Ghahremanine- jad2, Afshin Danehkar3, Fahimeh Koohdar1 1 Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotech- nology, Shahid Beheshti University, Tehran, Iran 2 Department of Plant Sciences, Faculty of Biological Sciences, Kharazmi University, Teh- ran, Iran 3 Department of Environmental Sciences, Faculty of Natural Resources, University of Teh- ran, Karaj, Iran *Corresponding author. E-mail: msheidai@sbu.ac.ir Abstract. The genus Avicennia contains of 8 species which show a great extent of mor- phological and genetic variability, which make taxonomy of the genus difficult. Molec- ular barcoding along with advancement in computational approaches may be proper methods to investigate and assess the efficiency of different molecular genetic regions in Avicennia species delineation and also produce data on species evolution and diver- gence. The aims of present study were to utilize multiple genetic data for the species delineation and study the phylogeny of the genus. Moreover, we developed a hypoth- esis on biogeography of these species with respect to barcode divergence. The results showed that both Internal transcribed spacer (ITS) and trnHG–psbA intergenic spacer (trnHG-psbA) sequences may be used in Avicennia species delineation. Barcode gap analysis and nucleotide difference of the studied taxa showed significant Fst for pair- wise species comparison and the role of nucleotide changes in Avicennia speciation. Keywords: Avicennia, Barcode-gap analysis, Genetic differentiation, Nucleotide differ- ence speciation. INTRODUCTION DNA barcoding is applied to plant and animal species with the aim to improve organismal identification and taxonomic clarification. The main principles of DNA barcoding are standardization, minimalism, and scalabil- ity, which means selection one or a few standard loci that can be sequenced routinely and reliably in very large and diverse sample sets, and obtaining a reliable and conveniently comparable data to differentiate the species in question from one another (Hollingsworth et al. 2011). Controversy exits on the use and choosing the plant molecular barcode markers. Different researches resulted in general agreement that several dif- ferent marker combinations produce equivalent performance, and that none of the proposed barcodes is perfect in every respect (Seberg and Petersen 4 Laleh Malekmohammadi et al. 2009). Utilizing a multiple approach for a better species differentiation has been suggested by several authors (see for example, Fazekas et al. 2008). In most of the studies, researchers use of a common, easily amplified and aligned region such as rbcL, trn L-F spacer regions, mat K, trnHG-psbA, nrITS1, nrITS2 or the full ITS1-5.8S-ITS2 (nrITS), as suggested by the CBOL Plant Working Group and BOLD (Cbol 2009; Ratnasingham and Hebert 2007). The genus  Avicennia is composed of eight species of mangrove trees which grow in intertidal zones in tropi- cal and temperate regions of the world. These plant spe- cies are economically important as they are extensively used as medicinal plants. In fact, different parts of these plants have ethno medicinal applications for treatment of various diseases such as cancer, diabetes, malaria, rheumatism, asthma, small pox and ulcer (Hrudayanath et al. 2016). These species show variation and are taxo- nomically complex due to vast geographical distribution and introgressive hybridization (Mori et al. 2015). There- fore, the aims of present study are: 1- Assessment of dif- ferent molecular markers in Avicennia species delinea- tion through barcode analysis, 2- Species relationships based on molecular markers, and 3- Biogeographi- cal distribution of these species with regard to DNA sequence divergence. MATERIALS AND METHODS In this study, we used published data on trn L-F, trn- HG-psbA and ITS sequences for a number of Avicennia species which are reported from different parts of the world in NCBI site (Table 1, 2 and 3). Data analyses DNA sequences obtained were initially aligned by MUSCLE program and cured accordingly. The total length, polymorphic sites, average of p distance, Genetic diversity within the studied species, the Fst values for the sequences and Maximum Liklihod phylogenetic tree based on these sequences as well as Tajimas’D test was performed as implemented in MEGA ver. 7 (Kumar et al. 2016). Mantel test performed with 1000 permutations, for showing significant association between nucleotide difference of the studied species and population to the geographical longitude and altitude. The range of Bayes- ian probability value obtained for species with Mr. Bayes analysis (Ronquist et al. 2012). Barcoding analysis and windows sliding of nucleotides were performed by Bar- codingR, and spider package, while Skyline plot and mismatch distribution of nucleotides were determined by ape, and pegas package in R. Monophyletic phyloge- netic tree and its statistical test of Rosenberg (2007), as implemented in R package. We used RASP (Reconstruc- tion of ancestral states in phylogeny) program ver. 4.2 in the RASP-Bayesian analyzed phylogenetic tree. RESULTS Species delimitation and barcode gap analysis We used published data on trn L-F, trnHG-psbA and ITS sequences for a number of Avicennia species which are reported from different parts of the world (Table 1). In the first step, we evaluated the efficiency of these molecular markers in species delineation, and finally, we extracted different evolutionary information from the one with the highest degree of efficiency. Details of the studied sequences with regard to Avi- cennia species differentiation and species phylogeny are presented below. Available data on trn L-F sequence is very limited and is available for only 21 samples of Avi- cennia officinalis, A. marina, and A. alba. These sequenc- es had a total length 296 bp, with only 14 polymorphic sites, and average p distacne = 0.006. The ML phyloge- Table 1. Voucher information and GenBank accession numbers of taxa sampled for the genus Avicennia based on trnL-F data. Sp Accession number Avicennia officinalis KT074999.1 Avicennia officinalis MH215695.1 Avicennia officinalis MH215694.1 Avicennia officinalis MH215693.1 Avicennia officinalis MH215692.1 Avicennia officinalis MH215691.1 Avicennia officinalis MH215690.1 Avicennia officinalis MH215689.1 Avicennia alba MH215683.1 Avicennia alba MH215682.1 Avicennia alba MH215681.1 Avicennia alba MH215680.1 Avicennia alba MH215679.1 Avicennia marina KT074998.1 Avicennia marina MH215688.1 Avicennia marina MH215687.1 Avicennia marina MH215686.1 Avicennia marina MH215685.1 Avicennia marina MH215684.1 Avicennia marina KM888791.1 Avicennia marina JQ728990.1 5Avicennia genus molecular phylogeny and barcoding: A multiple approach netic tree based on these sequences (Fig. 1), revealed that only the samples of A. alba can be differentiated from the other two species. Analyses performed by Bayesian method of spe- cies barcoding as implemented in Barcoding package in R program indicated that only 25% of the studied sam- ples have success in species identification, and the oth- ers may not be differentiated. In this analysis also the higher degree of Bayesian probability was obtained for A. alba (0.50-0.97). The probability value obtained for the other species was about 0.06 only. Sliding Windows of the mini-barcodes within trn L-F barcode sequence 9 (Fig. 2), also showed genetic distance in mini-barcodes between A. alba and the other species studied. TrnHG-psbA sequence efficiency in species delineation and barcoding TrnHG- psbA sequence data is available for 26 sam- ple in Avicennia species (Table 2) namely, A. officinalis, A. marina, A. bicolor, A. germinans, A. alba, and A. rumphiana. These samples have also been reported from different parts of the world. Preliminary analysis of trnHG- psbA sequences indi- cated that the total length of the studied sequences is 167 bp, with 52 polymorphic sites, and average p distance = 0.09. Table2 . Voucher information and GenBank accession numbers of taxa sampled for the genus Avicennia based on trnHG-psbA data. Sp Accession number Avicennia officinalis KT161361.1 Avicennia officinalis MN117565.1 Avicennia officinalis MN117564.1 Avicennia officinalis MN117563.1 Avicennia officinalis MN117562.1 Avicennia officinalis MN117561.1 Avicennia officinalis MN117560.1 Avicennia officinalis MN117559.1 Avicennia alba MN117553.1 Avicennia alba MN117552.1 Avicennia alba MN117551.1 Avicennia alba MN117550.1 Avicennia alba MN117549.1 Avicennia alba JX448690.1 Avicennia marina KT161360.1 Avicennia marina MN117558.1 Avicennia marina MN117557.1 Avicennia marina MN117556.1 Avicennia marina MN117555.1 Avicennia marina MN117554.1 Avicennia marina JX448688.1 Avicennia germinans KC420634.1 Avicennia germinans KJ426610.1 Avicennia germinans HG963703.1 Avicennia bicolor KC420633.1 Avicennia rumphiana JX448689.1 Figure 1. ML Phylogenetic tree of Avicennia species based on ITS sequences. Figure 2. Mini-barcode sliding windows of trnL-F sequence in Avi- cennia species, showing genetic distance of Avicennia alba with the other studied taxa. 6 Laleh Malekmohammadi et al. Table 3. Voucher information and GenBank accession numbers of taxa sampled for the genus Avicennia based on ITS data. Sp Accession number Avicennia alba EF540977.1 Avicennia alba AF365980.1 Avicennia alba MH243937.1 Avicennia alba MH243936.1 Avicennia alba MH243935.1 Avicennia alba MH243934.1 Avicennia alba MG880036.1 Avicennia alba MG880035.1 Avicennia alba MG880034.1 Avicennia alba MG880033.1 Avicennia alba MG880032.1 Avicennia alba MG880031.1 Avicennia alba MG880030.1 Avicennia alba MG880029.1 Avicennia alba MG880028.1 Avicennia alba EU528876.1 Avicennia alba KX641594.1 Avicennia alba KJ784551.1 Avicennia alba KF848261.1 Avicennia bicolor EF540988.1 Avicennia bicolor EF540988.1 Avicennia bicolor EF540987.1 Avicennia bicolor EU352151.1 Avicennia bicolor EU352150.1 Avicennia bicolor EU352149.1 Avicennia bicolor AF365977.1 Avicennia bicolor EU528877.1 Avicennia germinans EF540990.1 Avicennia germinans EF540985.1 Avicennia germinans EF540984.1 Avicennia germinans EF540983.1 Avicennia germinans EF540982.1 Avicennia germinans EF540981.1 Avicennia germinans EF540980.1 Avicennia germinans EU352146.1 Avicennia germinans EU352147.1 Avicennia germinans KX641596.1 Avicennia germinans MG880047.1 Avicennia germinans MG880046.1 Avicennia germinans MG880045.1 Avicennia germinans MG880041.1 Avicennia germinans MG880040.1 Avicennia germinans MG880039.1 Avicennia germinans MG880038.1 Avicennia germinans MG880037.1 Avicennia germinans DQ469846.1 Avicennia germinans DQ469845.1 Avicennia germinans DQ469860.1 Avicennia germinans DQ469859.1 Sp Accession number Avicennia germinans DQ469858.1 Avicennia germinans DQ469857.1 Avicennia germinans DQ469856.1 Avicennia germinans DQ469855.1 Avicennia germinans DQ469854.1 Avicennia germinans DQ469853.1 Avicennia germinans DQ469852.1 Avicennia integra KX641598.1 Avicennia officinalis MH243949.1 Avicennia officinalis MH243948.1 Avicennia officinalis MH243947.1 Avicennia officinalis MH243946.1 Avicennia officinalis MH243945.1 Avicennia officinalis MH243944.1 Avicennia officinalis MH243943.1 Avicennia officinalis MG880054.1 Avicennia officinalis MG880053.1 Avicennia officinalis MG880052.1 Avicennia officinalis MG880051.1 Avicennia officinalis MG880050.1 Avicennia officinalis KX641597.1 Avicennia officinalis KJ784553.1 Avicennia officinalis KF848263.1 Avicennia rumphiana KX641595.1 Avicennia schaueriana EF540986.1 Avicennia schaueriana DQ469862.1 Avicennia schaueriana AB861412.1 Avicennia schaueriana AB861385.1 Avicennia schaueriana AB861382.1 Avicennia schaueriana AB861365.1 Avicennia schaueriana AB861357.1 Avicennia schaueriana AB861354.1 Avicennia schaueriana AB861345.1 Avicennia schaueriana AB861327.1 Avicennia schaueriana AB861326.1 Avicennia schaueriana AB861325.1 Avicennia schaueriana AB861307.1 Avicennia schaueriana AB861306.1 Avicennia schaueriana AB861305.1 Avicennia schaueriana AB861287.1 Avicennia schaueriana AB861286.1 Avicennia schaueriana AB861285.1 Avicennia schaueriana AB861284.1 Avicennia schaueriana AB861280.1 Avicennia schaueriana AB861270.1 Avicennia schaueriana AB861266.1 Avicennia schaueriana AB861265.1 Avicennia schaueriana AB861263.1 Avicennia schaueriana AB861257.1 Avicennia schaueriana AB861251.1 Avicennia schaueriana AB861246.1 7Avicennia genus molecular phylogeny and barcoding: A multiple approach Bayesian method analysis of barcoding for trnHG- psbA sequences, reveals that about 53% of the samples are identified with success. Bayesian probability value obtained for A. officinalis samples, ranged from 0.24 to 0.96. The same value for A. germinans, ranged from 0.5- 0.95, for A. marina and A. alba the value ranged from was 0.2 to 0.96. Monophyletic phylogenetic tree and its statistical test of Rosenberg (2007), as implemented in R package is presented in Fig. 3. The red circles on the nodes of this phylogenetic tree indicates that monophyly has passed the significant test at p = 0.05. Therefore, we observe the presence of significant monophyletic groups for some of the sample in A. officinalis, A. alba, A. germinans, and A. marina. We have also some cases of admixture between the studied species which makes that clade non- monophyletic. TrnHG- psbA nucleotide difference in the studied Avicennia species is presented in Fig. 4. This plot shows a great difference of the trnHG- psbA nucleotides, which is a significant difference according to chi-square and Snn test of Hudson (Hudson, 2000). The Fst values for trnHG- psbA sequences in the stud- ied species ranged from 0.40-0.65. Inter-specific genetic differentiation estimates obtained for the trnHG- psbA nucleotide produced chi-square = 63.321, with P-value = 0.0012. Similarly, Hudson Snn after 1000 permutations produced Snn = 0.86, P<0.01. These values indicate signif- icant difference among the studied samples. Pair-wise mismatch plot of these sequences (Fig. 5), revealed that, almost all the studied species-pairs differ significantly in their trnHG- psbA nucleotides. Sp Accession number Avicennia schaueriana AB861245.1 Avicennia schaueriana AB861244.1 Avicennia schaueriana AB861240.1 Avicennia schaueriana AB861231.1 Avicennia schaueriana AB861226.1 Avicennia schaueriana AB861225.1 Avicennia schaueriana AB861224.1 Avicennia schaueriana AB861222.1 Avicennia schaueriana AB861220.1 Avicennia marina MF063712.1 Avicennia marina MF063711.1 Avicennia marina MF063710.1 Avicennia marina MF063709.1 Avicennia marina MF063708.1 Avicennia marina EF540978.1 Avicennia marina AF477771.1 Avicennia marina AF477770.1 Avicennia marina MN883387.1 Avicennia marina MN883386.1 Avicennia marina MN883385.1 Avicennia marina MN883384.1 Avicennia marina MH243942.1 Avicennia marina MH243941.1 Avicennia marina MH243940.1 Avicennia marina MH243939.1 Avicennia marina MH243938.1 Avicennia marina MG880049.1 Avicennia marina MG880048.1 Avicennia marina MK027295.1 Avicennia marina EU528879.1 Avicennia marina KM652500.1 Avicennia marina KF848262.1 Avicennia marina DQ469861.1 Avicennia marina subsp. marina KX641593.1 Avicennia marina subsp. eucalyptifolia KX641592. Avicennia marina subsp. australasica KX641591. Avicennia marina subsp. australasica AF365978.1 Figure 3. Monophyly analysis of Avicennia species based on trn- HG-psbA sequences. aThe red circles on the nodes indicate that the clade is significant at p = 0.05. Figure 4. trnHG-psbA nucleotide difference among Avicennia spe- cies. 8 Laleh Malekmohammadi et al. Mini-barcode analysis by windows sliding (Fig. 6), also showed that trnHG- psbA sequences contain mini-bar- codes which differs among Avicennia species. Therefore, trnHG- psbA sequences could be utilized for Avicennia. species delineation. Mini-barcode analysis by windows sliding (Fig. 7), also showed that trnHG- psbA sequences contain mini- barcodes which differs among Avicennia species. There- fore, trnHG- psbA sequences could be utilized for Avi- cennia species delineation. ITS sequences species delineation and barcoding ITS sequences (Table3) obtained had total length 494 bp, with polymorphic sites = 88, and average p dist = 0.038. Bayesian method analysis revealed that about 42% of the samples are identified correctly. Bayes- ian probability value obtained for Avicennia alba ranged from 0.12 to 0.99, for A. bicolor ranged from 0.20 to 0.99, and for A. germinans, ranged from 0.16 to 1.00. Almost the same ranges were obtained for other species. ML phylogenetic tree of 135 species samples (Fig. 8), pro- Figure 5. Pair-wise mismatch plot of trnHG-psbA sequences in Avi- cennia species. Figure 6. Window sliding of trnHG-psbA sequences in Avicennia species, showing that these species differ in mini-barcodes. Figure 7. TrnHG-psbA sequences in Avicennia species, showing mini-barcodes which differentiate the studied taxa. Figure 8. ML Phylogenetic tree of Avicennia species based on ITS sequences. 9Avicennia genus molecular phylogeny and barcoding: A multiple approach duced almost distinct separate clades for the studied species, but some degree of admixtures was observed too. For the test of monophyly, we kept randomly some of the replicates of each species. The result of monophyly and its statistical test of Rosenberg is presented in Fig. 9. The red circles on some of the tree nodes indicates the monophyletic clade which is significant at p = 0.05. We also obtained monophyletic clades for some of the sample in A. officinalis, A. alba, A. germinans, A. bicolor and A. marina. We have also some cases of admixture between the studied species which makes some of the clade non-monophyletic. The nucleotide difference in ITS sequences of the studied Avicennia species is presented in Fig. 10. The plot shows a great difference, which is a significant dif- ference according to chi-square and Snn test of Hudson. The Fst values for ITS sequences ranged from 0.43- 0.99. The inter-specific genetic differentiation estimates obtained for the ITS nucleotide produced chi-square = 90.26, with P-value = 0.001. Similarly, Hudson Snn after 1000 permutations produced Snn = 0.82, P<0.001. These values indicate significant difference among the stud- ied samples. Mismatch plot of ITS sequences (Fig. 11), revealed that, almost all the studied species-pairs differ significantly in their trnHG- psbA nucleotides. Moreover, Tajimas’D obtained was 0.32, which indicates the pres- ence of a positive selection on ITS sequences and there- fore ITS changes may be in some way related to specia- tion events in the genus Avicennia. Windows sliding of ITS sequences also revealed the occurrence of mini-barcodes in these sequences which also differed greatly among the studied species. For example, barcode sequences in some of the species are provided in Figs 12 and 13. Genetic diversity within the studied species ranged from 0.03 in A. alba to 0.12 in A. germinans, while inter- specific genetic distance, 0.04 between A. alba and A. officinalis, and 0.08 between A. marina and A. integra, to 0.43, between A. germinans and A. alba. DNA barcoding gap analysis of ITS sequences is presented in Fig. 14. Both intra- and inter-specific sequence gaps, supports the use of ITS sequences for delineation of Avicennia species. Mantel test performed with 1000 permutations, showed no significant association between nucleotide difference of the studied species and population to the geographical longitude and altitude (Correlation r = 0.044, p = 0.1456). Details of Avicennia species diversification based on ITS sequences and in relation with geographical distri- Figure 9. Monophyly test of the studied Avicennia species based on ITS sequences. aThe red circles on the. nodes indicate a significant monophyletic clade. Figure 10. The nucleotide difference in ITS sequences among Avi- cennia species. Figure 11. Mismatch plot of ITS sequences in Avicennia species. Figure 12. ITS Barcode sequences differentiating Avicennia alba and A. bicolor. 10 Laleh Malekmohammadi et al. bution of these species is presented in the RASP-Bayesi- an analyzed phylogenetic tree (Fig. 15). Two main clades are present in this phylogenetic tree. The species of A. alba, A. officinalis, A. marina, A. rumphiana, and A. integra, comprised the first major clade, while, A. schueriana, A. germinnas, and A. bicolor formed the second major clade. Looking at details of each major clade points out some interesting results. For example, most of A. marina samples were grouped together due to sequence similar- ity. Though Mantel test revealed no association between nucleotide difference and geographical longitude and lat- itude of the studied taxa, some interesting relationships between A. marina geographical populations can be seen Figure 13. Barcode ITS sequences differentiating Avicennia alba and A. germinans. Figure 14. Barcode gap analysis of ITS sequences revealing both intra- as well as inter-specific sequence difference in Avicennia spe- cies studied. Figure 15. RASP Bayesian tree of ITS data, placing the species studied in two major clades. 11Avicennia genus molecular phylogeny and barcoding: A multiple approach when we plot these studied specimens on the world map. For example, A. marina specimens studied from Saudi- Arabia and Egypt show sequence affinity and are placed close to each other in the phylogenetic tree. Moreover, the specimens studied from Madagascar, is placed close to the above said specimens. Madagascar is some-what close to and connected by Indian ocean to Saudi-Arabia and Egypt. Similarly, Avicennia marina samples from China and Australia, also show sequence similarity and form a separate sub-clade from the other specimens studied. The studied specimens from India stand in a separate sub-clade, far from the sub-clade of China-Australia. If we consider all the samples studied, biogeographi- cal distribution reveals that the species of A. marina, A. alba, and A. officinalis are mostly found in Asia and Australia region (Denoted A. in Fig. 16), while A. ter- minals, A. bicolor, and A. schauriena, are distributed in South America (Denoted B in Fig. 16). This may indicate speciation events in Avicennia in two different regions of the world. We have also provided barcodes for geo- graphical regions A and B, which shows nucleotide changes possibly associated / or the outcome of specia- tion in these two areas (Fig 16). DISCUSSION The present study showed that based on ITS sequence analysis, A. alba and A. officinalis show close affinity and comprise sister-clades. A. marina joins these two with some distance. A. germinans samples form three separate clades, which indicates a potential presence of infra-specific tax- on rank within this species. This is in accordance with earlier consideration which propose three different vari- Figure 16. Biogeographical distribution of Avicennia marina populations based on ITS sequences. 12 Laleh Malekmohammadi et al. eties for this species, which were later on were merged into a single species with no variety. A. bicolor showed close relationship to one of the clades of A. germinans. Sample of A. schaueriana com- prise a single distinct clade based on ITS data. However, the species relationships were partly dis- torted by trnHG- psbA sequence data. The three spe- cies of A. officinalis, A. marina, and A. alba, were placed inter-mixed to some degree. Close affinity bet ween A. germinans and A. bicolor are similar to ITS results. The close affinity between A. officinalis, A. marina, was also indicated by Li et al. (2016). These authors showed closer relationship between A.  rumphiana and  A. alba, which in agreement with our trnHG- psbA tree, and to some extent also with ITS-baes phylogenetic tree. According to Li et al. (2016), even though the first fossil record of  Avicennia in the IWP region dates back to the late Eocene of southwest Australia,  Avicennia spe- ciation was active during the Miocene. Similarly, they suggest that distribution of ancestral  Avicennia was like- ly to have been similar to its present location, extending from Japan to Borneo and from the Marshall Islands to the Red Sea. Therefore, the materials studied in this project, may have migrated from Japan, china or India, through red sea and reached to the countries like Iran, Egypt, and Sauidi-Arabia. In these migration path, Avi- cennia speciation may have resulted in formation of A. marina, A. officinalis and A. alba, as well as A. integra, and A. rumpiana. If we consider our ITS-based phylogenetic tree, we observe the species of the region B, viz. A. bicolor, A. germinans, A. schaueriana. are related through A. rumpiana and A. integra, to the species of region A. Therefore, we may suggest a preliminary hypothesis that through migration of either or both of A. rumpiana and A. integra, new speciation events resulted in the forma- tion of other species found in South America countries. We believe that more works are required to second this raw and immature hypothesis. Mangroves in general, have a broad distributional patterns, ability in long-distance dispersal and can adapt to rigorous environmental constraints associated with regular seawater inundation. However, the present day distributions of individual taxa show several instances of finite dispersal limitations, especially across open water. These dis-continuities, in the absence of current disper- sal barriers, may be explained by persistent past barriers (Duke et al. 2002). In present study we report genetic diversity both with the studied Avicennia species and between these taxa. A high levels of genetic diversity were also reported among the central populations of many mangrove spe- cies including Avicennia in the Indo-West Pacific (IWP) (Mantiquilla et al. 2021). Mori et al. (2015), suggest that A. bicolor, A. germinans  and  A. schaueriana  are three evolutionary lineages that present historical and ongo- ing hybridization. They also consider gene flow between A. germinans,  and  A. schaueriana by propagules rather than pollen in A. schaueriana. We also reported distinct inter-specific genetic dis- tance and significant Fst value among different spe- cies both within those species distributed in the A geo- graphical region (Australia, India, China), and those distributed in the B region (South America in general). In a similar investigation performed presence of a strong genetic structuring resulting in divergence among man- grove populations of Indian Ocean and South China Sea, as well as between South China Sea and Southwest- ern Pacific was reported (Mantiquilla et al. 2021). CONCLUSION With regard to Avicennia species taxonomy and the presence of high level of genetic diversity within these species, we provided distinct molecular barcodes for species delineation. We suggest it is suitable to utilize a combination of ITS nuclear sequences along with trn- HG- psbA spacer region of chloroplast genome for taxo- nomic purpose. LIST OF ABBREVIATIONS ITS: Internal transcribed spacer ML: Maximum Liklihod RASP: Reconstruction of ancestral states in phylogeny MEGA: Molecular Evolutionary Genetics Analysis ACKNOWLEDGEMENTS We thank the Iran National Science Foundation (INSF), for partial financial supportof this project (No.4002299). AUTHOR CONTRIBUTION STATEMENT Masood Sheidai: conceptualization of the project and corresponding author, Farrokh Ghahremaninejad: conceptualization of the project and data collection, Afshin Danehkar: conceptualization of the project and plant collection, Fahimeh Koohdar: conceptualization of the project and lab work, Laleh malekmohammadi: data collection and lab work and data analysis. 13Avicennia genus molecular phylogeny and barcoding: A multiple approach REFERENCES CBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Natl Acad Sci 106, 12794±12797 Fazekas AJ, Burgess KS, Kesanakurti PR, Graham SW, Newmaster SG, Husband BC, Percy DM, Hajibabaei M, BarrettScH (2008) Multiple multilocus DNA bar- codes from the plastid genome discriminate plant species equally well. PLoS ONE 3: e2802. Hollingsworth PM, Graham SW, Little DP (2011) Choos- ing and Using a Plant DNA Barcode. PLoS ONE 6(5): e19254 Hudson RR (2000) A new statistic for detecting genetic differentiation. 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